Dendrimer conjugates

ABSTRACT

The present invention relates to novel therapeutic and diagnostic dendrimers. In particular, the present invention is directed to dendrimer-linker conjugates, methods of synthesizing the same, compositions comprising the conjugates, as well as systems and methods utilizing the conjugates (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutics, imaging, and/or targeting agents (e.g., in disease (e.g., cancer) diagnosis and/or therapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugates of the present invention may further comprise one or more components for targeting, imaging, sensing, and/or providing a therapeutic or diagnostic material and/or monitoring response to therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional PatentApplication No. 61/101,461, filed Sep. 30, 2008, hereby incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract No.5RO1CA119409 awarded by the National Institutes of Health, and ContractNo. W911NF-07-1-0437 awarded by Defense Advanced Research ProjectsAgency. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel therapeutic and diagnosticdendrimers. In particular, the present invention is directed todendrimer-linker conjugates, methods of synthesizing the same,compositions comprising the conjugates, as well as systems and methodsutilizing the conjugates (e.g., in diagnostic and/or therapeuticsettings (e.g., for the delivery of therapeutics, imaging, and/ortargeting agents (e.g., in disease (e.g., cancer) diagnosis and/ortherapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugatesof the present invention may further comprise one or more components fortargeting, imaging, sensing, and/or providing a therapeutic ordiagnostic material and/or monitoring response to therapy.

BACKGROUND OF THE INVENTION

Cancer remains the number two cause of mortality in the United States,resulting in over 500,000 deaths per year. Despite advances in detectionand treatment, cancer mortality remains high. New compositions andmethods for the imaging and treatment (e.g., therapeutic) of cancer mayhelp to reduce the rate of mortality associated with cancer.

Severe, chronic pain is observed a variety of subjects. For example,there exist large numbers of individuals with sever pain associated witharthritis, autoimmune disease, injury, cancer, and a host of otherconditions.

A vast number of different types of pain medications exist. For example,a number natural and synthetic alkaloids of opium (i.e., opioids) areuseful as analgesics for the treatment of severe pain. However, a numberof severe side effects associated with opioid and other pain medicationusage exist. For example, administration of opioid agonists oftenresults in intestinal dysfunction due to action of the opioid agonistupon the large number of receptors in the intestinal wall. Opioids aregenerally known to cause nausea and vomiting as well as inhibition ofnormal propulsive gastrointestinal function in animals, resulting inside effects such as constipation.

Pain medication (e.g., opioid)-induced side effects are a seriousproblem for patients being administered pain medications (e.g., opioidanalgesics) for both short term and long term pain management. Forinstance, more than 250,000 terminal cancer patients each year takeopioids, such as morphine, for pain relief, and about half of thosepatients experience severe constipation. At present, patients receivingopioid pain medications face the difficult choice of sufferingburdensome adverse effects (e.g., constipation) or ineffectiveanalgesia.

There exists a need for compositions, methods and systems for deliveringagents (e.g., diagnostic and/or therapeutic (e.g., cancer and/or paintherapeutics) to subjects that provide effective therapy (e.g., diseasetreatment, symptom relief, etc.) with reduced or eliminated sideeffects, even when administered in high doses.

SUMMARY

Battlefield trauma covers a range of injuries caused by differentmechanisms resulting at times in severe disturbances of vital functions,disability, fear and pain (see, e.g., Conventional warfare: ballistic,blast, and burn injuries. 1990, Department of the Army, Office of theSurgeon General, Borden Institute. 396; herein incorporated by referencein its entirety). Depending on the military situation, initial reliancefor first aid must either be administered by oneself, other troops orfield medics. It is not until the injured reach a Forward Surgical Team(FST) or a Battalion Aid Station (BAS) that trained personnel canadminister traditional pain and anxiolytic medications due to the needfor frequent assessment of the medications' side effects and need forsupportive measures to prevent drug-induced deterioration of vitalfunctions (see, e.g., Anesthesia and perioperative care of the combatcasualty. 1995, Department of the Army, Office of the Surgeon General,Borden Institute. 931; herein incorporated by reference in itsentirety). Unfortunately, the time for an injured soldier to arrive at aFST or BAS unit can be delayed for hours to days. Adequate pain therapymay be markedly delayed resulting in problems such as long term,psychological-psychiatric effects, chronic pain syndromes and PostTraumatic Stress Disorders. This prolonged exposure of service membersto inadequately treated pain is associated with considerable cost to thesoldier and society (see, e.g., Bloodworth, D., Phys Med Rehabil Clin NAm, 2006. 17(2): p. 355-79; herein incorporated by reference in itsentirety).

If one were to attempt to administer potent pain and anxiolytic/amnesticmedications to injured soldiers on the battlefield, it is important tounderstand the compensatory physiological responses required to preservevital functions during trauma and evaluate how these medications' sideeffects could potentiate combat-induced pathophysiology. Ventilationwith oxygenation and perfusion of vital organs—the heart, lungs andbrain—must be preserved in the face of major injuries and blood loss,while at the same time perfusion of other organ systems may virtuallyshut down. Such a reduced flow state may be maintained for hours or daysdepending on the soldier's condition, environment and other factors.Disturbance of this delicate cardio respiratory balance by painmedications needs to be considered carefully lest it be detrimental oreven fatal to the injured. Reports of blood gasses of injured personneltaken at the time of admission to the FST unit illustrate the degree ofcardio, respiratory and metabolic compromise that occur in non-fatalinjuries. Approximately 35% of injured entrants had pH levels in therange of 7.0 to 7.20, and as low as 6.9 while pCO2 levels were often inthe 50-60 mmHg range and could be as high as 80-90 mmHg in severetrauma. Oxygen saturation levels were as low as 40% while the lowesthematocrit encountered was 9 mg/dl. This shows the dire nature of combatinjuries, and provides an insight into why the administration ofanalgesic and anxiolytic/amnestic medications with vasodilatory andrespiratory depressive side effects has been avoided. It also suggeststhe need for physiologically triggered feedback regulation to preventworsening of these derangements.

The unique conditions of battlefield trauma require a complex painrelief solution. For example, there is a need for a form of sustainedtherapeutic that could relieve pain over many hours or even days. Thissituation is further complicated by the need for battlefieldtherapeutics to be easily administered, preferably without the technicalchallenge of intravenous access or continuous administration. Therapyshould not require monitoring, since uninjured soldiers will be involvedin ongoing combat. Some effective pain medications (e.g., narcoticanalgesics) have serious limitations. For example, narcotic analgesicsare short acting, the therapeutic index is relatively narrow, and thedoses of drug that cause analgesia are not greatly separated from thosethat cause serious side effects, including respiratory depression andhypotension due to vasodilatation (see, e.g., Bloodworth, D., Phys MedRehabil Clin N Am, 2006. 17(2): p. 355-79; herein incorporated byreference in its entirety). Such side effects can actually worsen thephysiological derangements of acute trauma. Respiratory depression leadsto respiratory acidosis that can cause metabolic derangements. This canalso exacerbate metabolic acidosis due to traumatic injury, andsustained depression can lead to death from hypoxia due to respiratoryfailure. Hypotension can trigger shock and cardiovascular collapse,especially in an individual who has already suffered traumatic bloodloss. Thus, to provide autonomously effective narcotic analgesia overlong periods of time in the battlefield, a need exists for a sustainedrelease narcotic with a widened therapeutic index allowing for sustainedanalgesia in the absence of respiratory depression. This formulationshould also be easily administered (e.g., through intramuscularauto-injector). Finally, lack of sedation and an absence of systemicside effects are acutely important given the fact that individuals withtraumatic injuries in the battlefield may need to participate inself-extraction to safety.

The present invention provides compositions and related methodsaddressing such needs. In particular, the present invention providescompositions comprising dendrimer molecules (e.g., polyamideamine(PAMAM) dendrimers, polypropylamine (POPAM) dendrimers, or PAMAM-POPAMdendrimers) conjuguated to one or more pain relief agents (e.g., prodruganalgesic molecules, prodrug anxiolytic drugs, prodrug amnestic drugs).In some embodiments, the dendrimers conjugated to one or more painrelief agents are configured for controlled and/or sustained release ofthe pain relief agents (e.g., through use of targeting agents, linkingagents, and/or trigger agents conjugated to the dendrimer and/or painrelief agent). In some embodiments, the pain relief agent conjugated tothe dendrimer is active upon administration to a subject. In someembodiments, sustained release (e.g., slow release over a period of24-48 hours) of the pain relief drug is accomplished through conjugatingthe pain relief drug to the dendrimer through, for example, a linkageagent connected to a trigger agent that slowly degrades in a biologicalsystem (e.g., ester linkage). In some embodiments, constitutively activerelease of the pain relief drug is accomplished through conjugating thepain relief drug to the dendrimer through, for example, a linkage agentconnected to a trigger agent that renders the pain relief agentconstitutively active in a biological system (e.g., amide linkage, etherlinkage). In some embodiments, sustained release (e.g., a slow releasemechanism that achieves analgesic concentrations over a period of, forexample, 24-48 hours) of the pain relief agent prevents adverse sideeffects of the pain relief agent (e.g., respiratory failure, adversecardiovascular consequences).

In certain embodiments, the compositions comprising dendrimer moleculesconjugated to one or more pain relief agents are co-administered withadditional agents designed to prevent adverse side effects of painrelief agents (e.g., respiratory failure, adverse cardiovascularconsequences). In some embodiments, the compositions comprisingdendrimer molecules conjuguated to one or more pain relief agents (e.g.,narcotic prodrugs) are co-administered with a pain relief agentantagonist (e.g., a narcotic antagonist that is modulated to permitanalgesia while preventing respiratory depression). In some embodiments,a feedback system is employed so as to prevent respiratory failure andadverse cardiovascular consequences while still maintaining analgesia.In some embodiments, the “feedback” component is a rapid-acting narcoticantagonist (e.g., Naloxone) released only upon detection of symptoms ofrespiratory depression. In some embodiments, the biomarker used tomonitor respiratory depression and trigger the release of the antagonistis hypoxia (low pO2). Hypoxia is a sensitive and important marker as itis the direct cause of tissue injury from respiratory failure. It isalso more specific than lowered serum pH, a marker of respiratoryacidosis, since this is also observed in acute trauma situations as aresult of metabolic derangements. A fast acting antagonist released inresponse to hypoxia rapidly reverses respiratory depression, whichthereby increases the pO2, reversing the hypoxia and stopping therelease of the antagonist. In contrast, the narcotic itself wouldcontinue to be released at a slow and predictable rate. In this way,appropriate analgesia is achieved that is reversed only when absolutelynecessary to prevent respiratory failure. This would allow maintenanceof analgesia in the battlefield for prolonged periods of time withoutmonitoring of the wounded. In some embodiments, as shown in FIG. 32A,the present invention provides compositions comprising a plurality ofpain relief agents coupled to dendrimers with a linkage agent connectedto a trigger agent that slowly degrades in a biological system (e.g.,ester linkage) (as shown in FIG. 32A, the trigger agent is an ester bondthat is released by serum esterases to mediate sustained-releaseanalgesia). When administered together, the plurality of pain reliefagents (e.g., Ketamine and Lorazepam) have favorable analgesic andanxiolytic/amnestic qualities, and relatively broad therapeutic indexes.Compositions comprising a plurality of pain relief agents (e.g.,Ketamine and Lorazepam) provide analgesia without the cardiovasculareffects of opioids and minimize their major complication, that beingrespiratory depression. As a feedback mechanism, compositions of theinvention comprising a pain relief agent antagonist (e.g., Doxapram) arecomplexed with a dendrimer through charge interaction (e.g., that isreleasable by, for example, acidosis). While acidosis may be observedfrom causes other than respiratory depression, this feedback mechanismis unique in that Doxapram stimulates respirations without reducinganalgesia. As such, the present invention provides release of Doxapramregardless of the source of the acidosis (e.g., traumatic injuries,hemorrhagic shock, burns or rhabdomyolysis). Thus, the present inventionprovides a safe analgesia with easily achieved physiological feedback.

In some embodiments, targeting agents are conjugated to the dendrimersfor delivery of the dendrimers to desired body regions (e.g., to thecentral nervous system (CNS). The targeting agents are not limited totargeting specific body regions. In some embodiments, the targetingagents target the central nervous system (CNS). In some embodiments,targeting agents target the peripheral nervous system, specific nerves(e.g., perception nerves, pain nerves, pressure nerves, etc.), muscles,and/or tendons. In some embodiments, where the targeting agent isspecific for the CNS, the targeting agent is transferrin (see, e.g.,Daniels, T. R., et al., Clinical Immunology, 2006. 121(2): p. 159-176;Daniels, T. R., et al., Clinical Immunology, 2006. 121(2): p. 144-158;each herein incorporated by reference in their entireties). In someembodiments, the targeting agents target neurons within the centralnervous system (CNS). In some embodiments, where the targeting agent isspecific for neurons within the CNS, the targeting agent is a synthetictetanus toxin fragment (e.g., a 12 amino acid peptide (Tet 1)) (see,e.g., Liu, J. K., et al., Neurobiology of Disease, 2005. 19(3): p.407-418; herein incorporated by reference in its entirety). In someembodiments, locking agents designed to retain the dendrimer within aparticular body region are conjugated to the dendrimer (e.g., lockingagents designed to prevent back diffusion of a dendrimer across theblood brain barrier (BBB) (e.g., pyridinium molecule, which whenactivated by enzymatic reduction, becomes charged and locks thedendrimer in the CNS)). In some embodiments, trigger agents areconjugated to the dendrimers so as to permit a controlled release of aparticular agent (e.g., a narcotic and/or narcotic antagonist). Thedendrimers are not limited to particular types of trigger agents. Insome embodiments, sustained release (e.g., slow release over a period of24-48 hours) of the pain relief drug is accomplished through conjugatingthe pain relief drug to the dendrimer through, for example, a linkageagent connected to a trigger agent that slowly degrades in a biologicalsystem (e.g., ester linkage). In some embodiments, constitutively activerelease of the pain relief drug is accomplished through conjugating thepain relief drug to the dendrimer through, for example, a linkage agentconnected to a trigger agent that renders the pain relief agentconstitutively active in a biological system (e.g., amide linkage, etherlinkage). In some embodiments, the trigger agent is designed to permitrelease of the drug conjugated to the dendrimer in the presence of brainenzymes (e.g., the trigger agent indolequinone is reduced by brainenzymes such as, for example, diaphorase). In some embodiments, thetrigger agent is designed to permit release of the drug conjugated tothe dendrimer upon detection of reduced pO2 concentrations (e.g.,through use of a trigger agent that detects reduced pO2 levels (e.g., are-dox linker)). The use of a re-dox linker provides directphysiological feed back in order to prevent consequences ofopoid-induced respiratory depression (e.g., cerebral hypoxia).

FIG. 32B shows two dendrimer conjugates designed for pain management ina subject. One of the dendrimers is conjugated to a morphine drugthrough a linkage agent connected to a trigger agent (e.g., esterlinkage) permitting sustained release . The other dendrimer isconjugated to a morphine antagonist (e.g., Naloxone) through a linkageagent connected to a trigger agent (e.g., re-dox linker) permittingrelease of the morphine antagonist upon detection of reduced pO2 levels.Each of the dendrimers are targeted for CNS delivery through conjugationof targeting agents specific for the CNS (e.g., transferrin, a synthetictetanus toxin fragment). Each of the dendrimers are designed forretention within the CNS through conjugation of locking agents designedto prevent back diffusion of the dendrimer across the BBB (e.g.,pyridinium molecule, which when activated be enzymatic reduction,becomes charged and locks the dendrimer in the CNS).

These two characteristics differentiate the sustained release of anarcotic, and the feedback release of a narcotic antagonist. Forexample, in some embodiments, the narcotic is linked such that it wouldremain constitutively active while coupled to the polymer orcontinuously released over time (e.g., through triggering agentsdesigned to permit sustained release) to provide prolonged activity,whereas, in some embodiments, the antagonist is active when releasedduring hypoxia to prevent respiratory failure. In some embodiments, theantagonist and agonist are attached to two identical populations ofdendrimers in a very consistent manner, and administered together (orseparately) to form a single drug delivery system.

Accordingly, the present invention provides compositions, systems andmethods for treating and/or managing pain in a subject through use ofdendrimers conjugated to pain relief agents and/or pain relief agentantagonists. The following discussion describes individual componentparts of the dendrimer and methods of making and using the same in someembodiments of the present invention. To illustrate the design and useof the systems and compositions of the present invention, the discussionfocuses on specific embodiments of the use of the compositions in thetreatment and reduction of pain suffered by a subject. These specificembodiments are intended only to illustrate certain preferredembodiments of the present invention and are not intended to limit thescope thereof. For example, although some discussion of the presentinvention involves battlefield injuries, other types apply (e.g.,general trauma settings).

In certain embodiments, the present invention provides compositionscomprising a dendrimer linked to a moiety comprising a trigger agent, alinkage agent, a targeting agent, and at least one therapeutic agent,wherein the therapeutic agent is a pain relief agent designed to reduceand/or eliminate pain in a subject and/or a pain relief agentantagonist. The composition are not limited to particular dendrimers. Insome embodiments, the dendrimer is, for example, a polyamideamine(PAMAM) dendrimer, a polypropylamine (POPAM) dendrimer, and aPAMAM-POPAM dendrimer. The compositions are not limited to particularlinkage agents (e.g., a spacer comprising between 1 and 8 straight orbranched carbon chains). In some embodiments, the linkage agent issubstituted or unsubstituted straight or branched carbon chain. In someembodiments, straight or branched carbon chains are substituted withalkyls. In some embodiments, the dendrimers are acetylated.

The compositions are not limited to particular trigger agents. In someembodiments, the trigger agents are configured to delay release of thepain relief agent from the moiety (e.g., an ester bond). In someembodiments, the trigger agents are configured to constitutively releasethe therapeutic agent from the moiety (e.g., an amide bond, an etherbond). In some embodiments, the trigger agent is configured to releasethe therapeutic agent from the moiety under conditions of acidosis. Insome embodiments, the trigger agent is configured to release thetherapeutic agent from the moiety under conditions of hypoxia (e.g.,indoquinones, nitroheterocyles, and nitroimidazoles). In someembodiments, the trigger agent is configured to release the therapeuticagent from the moiety in the presence of a brain enzyme (e.g., thetrigger agent is indolequinone and the brain enzyme is diaphorase).

The compositions are not limited to particular targeting agents. In someembodiments, the targeting agent is configured to permit the compositionto cross the blood brain barrier (e.g., transferrin). In someembodiments, the targeting agent is configured to permit the compositionto bind with a neuron within the central nervous system (e.g., thetargeting agent is a synthetic tetanus toxin fragment (e.g., an aminoacid peptide fragment (e.g., HLNILSTLWKYR))).

In some embodiments, the moiety further comprises a locking agent. Thecompositions are not limited to particular locking agents. In someembodiments, the locking agent, upon activation, prevents transfer ofthe composition across the blood brain barrier. In some embodiments, thelocking agent is a pyridinium molecule which is activated by enzymesspecific to the central nervous system. In some embodiments, the lockingagent is a re-dox system. In some embodiments, the re-dox system is the1,4-dihydrotrigonelline⇄trigonelline (coffearine) re-dox system, whereinconversion of lipophilic 1,4-dihydro form (L) in vivo to the hydrophilicquaternary form (L⁺) by oxidation prevents the composition fromdiffusing across the blood brain barrier.

The compositions are not limited to particular pain relief agents. Insome embodiments, the pain relief agents include, but are not limitedto, analgesic drugs, anxiolytic drugs, anesthetic drugs, antipsychoticdrugs, hypnotic drugs, sedative drugs, and muscle relaxant drugs.

In some embodiments, the analgesic drugs include, but are not limitedto, non-steroidal anti-inflammatory drugs, COX-2 inhibitors, andopiates. In some embodiments, the non-steroidal anti-inflammatory drugsare selected from the group consisting of Acetylsalicylic acid(Aspirin), Amoxiprin, Benorylate/Benorilate, Choline magnesiumsalicylate, Diflunisal, Ethenzamide, Faislamine, Methyl salicylate,Magnesium salicylate, Salicyl salicylate, Salicylamide, arylalkanoicacids, Diclofenac, Aceclofenac, Acemethacin, Alclofenac, Bromfenac,Etodolac, Indometacin, Nabumetone, Oxametacin, Proglumetacin, Sulindac,Tolmetin, 2-arylpropionic acids, Ibuprofen, Alminoprofen, Benoxaprofen,Carprofen, Dexibuprofen, Dexketoprofen, Fenbufen, Fenoprofen,Flunoxaprofen, Flurbiprofen, Ibuproxam, Indoprofen, Ketoprofen,Ketorolac, Loxoprofen, Naproxen, Oxaprozin, Pirprofen, Suprofen,Tiaprofenic acid), N-arylanthranilic acids, Mefenamic acid, Flufenamicacid, Meclofenamic acid, Tolfenamic acid, pyrazolidine derivatives,Phenylbutazone, Ampyrone, Azapropazone, Clofezone, Kebuzone, Metamizole,Mofebutazone, Oxyphenbutazone, Phenazone, Sulfinpyrazone, oxicams,Piroxicam, Droxicam, Lornoxicam, Meloxicam, Tenoxicam, sulphonanilides,nimesulide, licofelone, and omega-3 fatty acids. In some embodiments,the COX-2 inhibitors are selected from the group consisting ofCelecoxib, Etoricoxib, Lumiracoxib, Parecoxib, Rofecoxib, andValdecoxib. In some embodiments, the opiate drugs are selected from thegroup consisting of natural opiates, alkaloids, morphine, codeine,thebaine, semi-synthetic opiates, hydromorphone, hydrocodone, oxycodone,oxymorphone, desomorphine, diacetylmorphine (Heroin), nicomorphine,dipropanoylmorphine, diamorphine, benzylmorphine, Buprenorphine,Nalbuphine, Pentazocine, meperidine, diamorphine, ethylmorphine, fullysynthetic opioids, fentanyl, pethidine, Oxycodone, Oxymorphone,methadone, tramadol, Butorphanol, Levorphanol, propoxyphene, endogenousopioid peptides, endorphins, enkephalins, dynorphins, and endomorphins.

In some embodiments, the anxiolytic drugs include, but are not limitedto, benzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide(Librium), Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam,Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam,oxazepam (Serax), temazepam (Restoril, Normison, Planum, Tenox, andTemaze, Triazolam, serotonin 1A agonists, Buspirone (BuSpar),barbituates, amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone), hydroxyzine,cannabidiol, valerian, kava (Kava Kava), chamomile, Kratom, Blue Lotusextracts, Sceletium tortuosum (kanna) and bacopa monniera.

In some embodiments, the anesthetic drugs include, but are not limitedto, local anesthetics, procaine, amethocaine, cocaine, lidocaine,prilocaine, bupivacaine, levobupivacaine, ropivacaine, dibucaine,inhaled anesthetics, Desflurane, Enflurane, Halothane, Isoflurane,Nitrous oxide, Sevoflurane, Xenon, intravenous anesthetics,Barbiturates, amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone)), Benzodiazepines,alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam,Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam,temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax),temazepam (Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,Etomidate, Ketamine, and Propofol.

In some embodiments, the antipsychotic drugs include, but are notlimited to, butyrophenones, haloperidol, phenothiazines, Chlorpromazine(Thorazine), Fluphenazine (Prolixin), Perphenazine (Trilafon),Prochlorperazine (Compazine), Thioridazine (Mellaril), Trifluoperazine(Stelazine), Mesoridazine, Promazine, Triflupromazine (Vesprin),Levomepromazine (Nozinan), Promethazine (Phenergan)), thioxanthenes,Chlorprothixene, Flupenthixol (Depixol and Fluanxol), Thiothixene(Navane), Zuclopenthixol (Clopixol & Acuphase)), clozapine, olanzapine,Risperidone (Risperdal), Quetiapine (Seroquel), Ziprasidone (Geodon),Amisulpride (Solian), Paliperidone (Invega), dopamine, bifeprunox,norclozapine (ACP-104), Aripiprazole (Abilify), Tetrabenazine, andCannabidiol.

In some embodiments, the hypnotic drugs include, but are not limited to,Barbiturates, Opioids, benzodiazepines, alprazolam, bromazepam(Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam,Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam,nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,nonbenzodiazepines, Zolpidem, Zaleplon, Zopiclone, Eszopiclone,antihistamines, Diphenhydramine, Doxylamine, Hydroxyzine, Promethazine,gamma-hydroxybutyric acid (Xyrem), Glutethimide, Chloral hydrate,Ethchlorvynol, Levomepromazine, Chlormethiazole, Melatonin, and Alcohol.

In some embodiments, the sedative drugs include, but are not limited to,barbituates, amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone), benzodiazepines,alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam,Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam,temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax),temazepam (Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,herbal sedatives, ashwagandha, catnip, kava (Piper methysticum),mandrake, marijuana, valerian, solvent sedatives, chloral hydrate(Noctec), diethyl ether (Ether), ethyl alcohol (alcoholic beverage),methyl trichloride (Chloroform), nonbenzodiazepine sedatives,eszopiclone (Lunesta), zaleplon (Sonata), zolpidem (Ambien), zopiclone(Imovane, Zimovane)), clomethiazole (clomethiazole),gamma-hydroxybutyrate (GHB), Thalidomide, ethchlorvynol (Placidyl),glutethimide (Doriden), ketamine (Ketalar, Ketaset), methaqualone(Sopor, Quaalude), methyprylon (Noludar), and ramelteon (Rozerem).

In some embodiments, the muscle relaxant drugs include, but are notlimited to, depolarizing muscle relaxants, Succinylcholine, short actingnon-depolarizing muscle relaxants, Mivacurium, Rapacuronium,intermediate acting non-depolarizing muscle relaxants, Atracurium,Cisatracurium, Rocuronium, Vecuronium, long acting non-depolarizingmuscle relaxants, Alcuronium, Doxacurium, Gallamine, Metocurine,Pancuronium, Pipecuronium, and d-Tubocurarine.

The compositions are not limited to particular pain relief agentantagonists. In some embodiments, the pain relief agent antagonistsinclude drugs that counter the effect of a pain relief agent (e.g., ananesthetic antagonist, an analgesic antagonist, a mood stabilizerantagonist, a psycholeptic drug antagonist, a psychoanaleptic drugantagonist, a sedative drug antagonist, a muscle relaxant drugantagonist, and a hypnotic drug antagonist). In some embodiments, painrelief agent antagonists include, but are not limited to, a respiratorystimulant, Doxapram, BIMU-8, CX-546, an opiod receptor antagonist,Naloxone, naltrexone, nalorphine, levallorphan, cyprodime, naltrindole,norbinaltorphimine, buprenorphine, a benzodiazepine antagonist,flumazenil, a non-depolarizing muscle relaxant antagonist, andneostigmine.

In some embodiments, the moiety comprises a plurality of therapeuticagents (e.g., 2, 3, 4, 5, 10, 15, 50, 100, at any desired ratio). Insome embodiments, the moeity comprises a plurality of pain relief agents(e.g., ketamine and lorazepam). In some embodiments, the therapeuticagent is a pain relief agent, wherein the pain relief agent is morphine.In some embodiments, the therapeutic agent is a pain relief agentantagonist, wherein the pain relief agent antagonist is Doxapram. Insome embodiments, the therapeutic agent is a pain relief agentantagonist, wherein the pain relief agent antagonist is Naloxone.

In certain embodiments, the present invention provides methods forreducing pain in a subject (e.g., cat, dog, human, monkey, ape, cow,etc.) comprising administering to the subject at least one compositioncomprising a dendrimer linked to a moiety comprising a trigger agent, alinkage agent, a targeting agent, and at least one therapeutic agent,wherein the therapeutic agent is a pain relief agent designed to reduceand/or eliminate pain in a subject and/or a pain relief agent antagonist(as described above).

In some embodiments, the two compositions are administered to thesubject such that one of the compositions comprises a pain relief agentand one of the compositions comprises a pain relief agent antagonist. Insuch embodiments, for example, the pain relief agent is morphine and thepain relief agent antagonist is Naloxone. In such embodiments, forexample, the pain relief agent is ketamine and/or lorazepam and the painrelief agent antagonist is Doxapram.

In some embodiments, the present invention provides methods for treatingcancer localized with a subject's central nervous system (e.g., brain)administering to the subject at least one composition comprising adendrimer linked to a moeity comprising a trigger agent (e.g., a triggeragent that is sensitive to (e.g., is cleaved by) hypoxia) (e.g., atrigger agent that is sensitive to (e.g., is cleaved by) tumorassociated enzymes), a linkage agent, a targeting agent configured tocross the blood brain barrier, and at least one therapeutic agentconfigured for treating cancer. In some embodiments, the dendrimers aredesigned for retention within the CNS through conjugation of lockingagents designed to prevent back diffusion of the dendrimer across theBBB (e.g., pyridinium molecule, which when activated by enzymaticreduction, becomes charged and locks the dendrimer in the CNS).

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 2 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 3 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 4 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 5 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 6 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 7 shows the release of a therapeutic compound from a dendrimerconjugate in one embodiment of the invention.

FIG. 8 shows the release of a therapeutic compound from a dendrimerconjugate in one embodiment of the invention.

FIG. 9 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 10 shows the release of a therapeutic compound from esterasesensitive linker-dendrimer conjugate in one embodiment of the invention(Scheme 1).

FIG. 11 shows examples of several (A, B, and C) elimination linkersdesigned for esterase triggered cleavage.

FIG. 12 shows the characterization of therapeutic compound release fromdendrimer conjugates of the present invention.

FIG. 13 shows dendrimer conjugate and methods of synthesizing the samein some embodiments of the invention.

FIG. 14 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 15 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 16 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 17 shows an example of a dendrimer conjugate designed forglucuronidase triggered cleavage in one embodiment of the presentinvention.

FIG. 18 shows an example of a dendrimer conjugate designed for hypoxiainduced activation in one embodiment of the present invention.

FIG. 19 shows that, in some embodiments, a heteroaromatic nitro compoundpresent in a dendrimer conjugate of the present invention is reduced toeither an amine or a hydroxylamine, thereby triggering the spontaneousrelease of a therapeutic agent/drug.

FIG. 20 depicts the activation of a dendrimer conjugate comprisingeither a 1,4 or a 1,6 elimination linker in embodiments of the presentinvention.

FIG. 21 shows that a spacer (R2) can be used to decrease sterichindrance in a dendrimer conjugate in some embodiments of the presentinvention.

FIG. 22 depicts a dendrimer conjugate comprising a cyclization basedlinker in some embodiments of the present invention.

FIG. 23 depicts cyclization based linkers in some embodiments of theinvention.

FIG. 24 depicts a linker utilized in a dendrimer conjugate in someembodiments of the present invention.

FIG. 25 shows branched self-elimination linkers utilized in a dendrimerconjugate in some embodiments of the present invention.

FIGS. 26A and B depicts dendrimer conjugates provided in someembodiments of the present invention.

FIG. 27 shows a dendrimer comprising a simple ester (top portion offigure) and a dendrimer conjugate comprising an elimination linker(e.g., a 1, 6, elimination linker/spacer as shown in the bottomportion).

FIG. 28 shows a dendrimer conjugate comprising hydroxycamptothecin insome embodiments of the invention.

FIG. 29 shows a synthesis scheme for generating a dendrimer comprising ahypoxia induced linker.

FIG. 30 shows a synthesis scheme for generating a dendrimer comprising ahypoxia induced linker.

FIG. 31 shows a diagram depicting a mechanism of release of atherapeutic agent from a dendrimer comprising a hypoxia sensitivelinker.

FIG. 32 shows two dendrimer conjugates.

FIG. 33 shows a schematic representation of CNS locking targeted drug.

FIG. 34 shows a strategy to analyze drug conjugated to the dendrimerscaffold with ester linkage using HPLC.

FIG. 35 shows structures of pain relief agents.

FIG. 36 shows structures of morphine pro-drugs.

FIG. 37 shows the structure of a Naloxone pro-drug where the length ofthe spacer was varied to produce 3 additional Naloxone pro-drugs.

FIG. 38 shows drug formulations.

FIG. 39 shows time-dependent release kinetics of Morphine compoundsincubated in fresh frozen plasma.

FIG. 40 shows time-dependent release kinetics of Naloxone pro-drugincubated in fresh frozen plasma under hypoxia.

FIG. 41 shows a schematic depicting the complexation of drug to G5 PAMAMdendrimer.

FIG. 42 shows a scheme depicting synthesis used to form pro-drugdendrimer conjugates.

FIG. 43 shows a schematic showing the initial loading and subsequentdecrease of drug concentration in blood due to distribution andelimination. Models are used to determine an appropriate mixture ofcompounds needed to maintain the desired drug concentration.

FIG. 44 shows in vitro sustained release of morphine using a morphinepro-drug.

FIG. 45 shows the release kinetics of free morphine from the prodrug inthe various plasma samples.

FIG. 46 shows that naloxone is released from an indolequinone basednaloxone prodrug only under low oxygen conditions.

FIG. 47 shows sustained release of morphine in the guinea pig model overa six hour period with prodrug A.

FIG. 48 shows in vivo studies with a guinea pig model demonstrating thatnaloxone is release from naloxone—pro-drug only under low oxygenconditions.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “antagonist” or “pain relief agent antagonist”refers to an agent able to counter the effect of a pain relief agentand/or the effect of a pain relief agent (e.g., respiratory distress,cardiovascular distress).

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass) but for whom aconfirmatory test (e.g., biopsy and/or histology) has not been done orfor whom the stage of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancerand is sometimes found to not have cancer.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention.

As used herein, the term “initial diagnosis” refers to a test result ofinitial cancer diagnosis that reveals the presence or absence ofcancerous cells (e.g., using a biopsy and histology).

As used herein, the term “identifying the risk of said tumormetastasizing” refers to the relative risk (e.g., the percent chance ora relative score) of a tumor metastasizing.

As used herein, the term “identifying the risk of said tumor recurring”refers to the relative risk (e.g., the percent chance or a relativescore) of a tumor recurring in the same organ as the original tumor.

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental expose, and previous incidents of cancer,preexisting non-cancer diseases, and lifestyle.

As used herein, the term “characterizing cancer in subject” refers tothe identification of one or more properties of a cancer sample in asubject, including but not limited to, the presence of benign,pre-cancerous or cancerous tissue and the stage of the cancer.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor, whether the tumor has spread to otherparts of the body and where the cancer has spread (e.g., within the sameorgan or region of the body or to another organ).

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer on asubject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

As used herein, the term “characterizing tissue in a subject” refers tothe identification of one or more properties of a tissue sample (e.g.,including but not limited to, the presence of cancerous tissue, thepresence of pre-cancerous tissue that is likely to become cancerous, andthe presence of cancerous tissue that is likely to metastasize.

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer (e.g., asdetermined by the diagnostic methods of the present invention) on asubject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

As used herein, the term “non-human animals” refers to all non-humananimals including, but not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

As used herein, the term “drug” is meant to include any molecule,molecular complex or substance administered to an organism fordiagnostic or therapeutic purposes, including medical imaging,monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical andprophylactic applications. The term “drug” is further meant to includeany such molecule, molecular complex or substance that is chemicallymodified and/or operatively attached to a biologic or biocompatiblestructure.

As used herein, the term “purified” or “to purify” or “compositionalpurity” refers to the removal of components (e.g., contaminants) from asample or the level of components (e.g., contaminants) within a sample.For example, unreacted moieties, degradation products, excess reactants,or byproducts are removed from a sample following a synthesis reactionor preparative method.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using screening methods known in the art.

As used herein, the term “NAALADase inhibitor” refers to any one of amultitude of inhibitors for the neuropeptidase NAALADase(N-acetylated-alpha linked acidic dipeptidase). Such inhibitors ofNAALADase have been well characterizied. For example, an inhibitor canbe selected from the group comprising, but not limited to, those foundin U.S. Pat. No. 6,011,021, herein incorporated by reference in itsentirety.

As used herein, the term “nanodevice” or “nanodevices” refer, generally,to compositions comprising dendrimers of the present invention. As such,a nanodevice may refer to a composition comprising a dendrimer and metalnanoparticles (e.g., iron oxide nanoparticles (e.g., poly(styrenesulfonate) (PSS)-coated iron oxide nanoparticles)) of the presentinvention that may contain one or more functional groups (e.g., atherapeutic agent) conjugated to the dendrimer. A nanodevice may alsorefer to a composition comprising two or more different dendrimers ofthe present invention.

As used herein, the term “degradable linkage,” when used in reference toa polymer (e.g., PEG-hRNase conjugate of the present invention), refersto a conjugate that comprises a physiologically cleavable linkage (e.g.,a linkage that can be hydrolyzed (e.g., in vivo) or otherwise reversed(e.g., via enzymatic cleavage). Such physiologically cleavable linkagesinclude, but are not limited to, ester, carbonate ester, carbamate,sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal linkages (See,e.g., U.S. Pat. No. 6,838,076, herein incorporated by reference in itsentirety). Similarly, the conjugate may comprise a cleavable linkagepresent in the linkage between the polymer and hRNase, or, may comprisea cleavable linkage present in the polymer itself (e.g., such that whencleaved, a small portion of the polymer remains on the hRNase molecule)(See, e.g., U.S. Pat. App. Nos. 20050158273 and 20050181449, each ofwhich is herein incorporated by reference in its entirety). For example,a PEG polymer comprising an ester linkage can be utilized forconjugation to hRNase to create a PEG-hRNase conjugate (See, e.g.,Kuzlowski et al., Biodrugs, 15, 419-429 (2001). A conjugate thatcomprises a degradable linkage of the present invention is capable ofgenerating hRNase that is free (e.g., completely or partially free) ofthe polymer (e.g., in vivo after hydrolysis of the linkage).

A “physiologically cleavable” or “hydrolysable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond(e.g., typically a covalent bond) that is substantially stable in water(i.e., does not undergo hydrolysis under physiological conditions to anyappreciable extent over an extended period of time). Examples ofhydrolytically stable linkages include, but are not limited to,carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides,urethanes, and the like.

As used herein, the term “click chemistry” refers to chemistry tailoredto generate substances quickly and reliably by joining small modularunits together (see, e.g., Kolb et al. (2001) Angewandte Chemie Intl.Ed. 40:2004-2011; Evans (2007) Australian J. Chem. 60:384-395; Carlmarket al. (2009) Chem. Soc. Rev. 38:352-362; each herein incorporated byreference in its entirety).

As used herein, the term “one-pot synthesis reaction” or equivalentsthereof, e.g., “1-pot”, “one pot”, etc., refers to a chemical synthesismethod in which all reactants are present in a single vessel. Reactantsmay be added simultaneously or sequentially, with no limitation as tothe duration of time elapsing between introduction of sequentially addedreactants.

As used herein, an “ester coupling agent” refers to a reagent that canfacilitate the formation of an ester bond between two reactants. Thepresent invention is not limited to any particular coupling agent oragents. Examples of coupling agents include but are not limited to2-chloro-1-methylpyridium iodide and 4-(dimethylamino)pyridine, ordicyclohexylcarbodiimide and 4-(dimethylamino)pyridine or diethylazodicarboxylate and triphenylphosphine or other carbodiimide couplingagent and 4-(dimethylamino)pyridine.

As used herein, the term “glycidolate” refers to the addition of a2,3-dihydroxylpropyl group to a reagent using glycidol as a reactant. Insome embodiments, the reagent to which the 2,3-dihydroxylpropyl groupsare added is a dendrimer. In some embodiments, the dendrimer is a PAMAMdendrimer. Glycidolation may be used generally to add terminal hydroxylfunctional groups to a reagent.

As used herein, the term “ligand” refers to any moiety covalentlyattached (e.g., conjugated) to a dendrimer branch; in preferredembodiments, such conjugation is indirect (e.g., an intervening moietyexists between the dendrimer branch and the ligand) rather than direct(e.g., no intervening moiety exists between the dendrimer branch and theligand). Indirect attachment of a ligand to a dendrimer may exist wherea scaffold compound (e.g., triazine scaffold) intervenes. In preferredembodiments, ligands have functional utility for specific applications,e.g., for therapeutic, targeting, imaging, or drug delivery function(s).The terms “ligand”, “conjugate”, and “functional group” may be usedinterchangeably.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel therapeutic and diagnosticdendrimers. In particular, the present invention is directed todendrimer-linker conjugates, methods of synthesizing the same,compositions comprising the conjugates, as well as systems and methodsutilizing the conjugates (e.g., in diagnostic and/or therapeuticsettings (e.g., for the delivery of therapeutics, imaging, and/ortargeting agents (e.g., in disease (e.g., cancer) diagnosis and/ortherapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugatesof the present invention may further comprise one or more components fortargeting, imaging, sensing, and/or providing a therapeutic ordiagnostic material and/or monitoring response to therapy.

Accordingly, in some embodiments, the present invention provides alinker conjugated to an agent (e.g., therapeutic agent, imaging agent,targeting agent, triggering agent) (e.g., that can be conjugated to adendrimer (e.g., for specific targeting and/or therapeutic use of thedendrimer)). Thus, in some embodiments, the present invention providesmethods of synthesizing dendrimer conjugates (e.g., PAMAM dendrimers)comprising linkers (e.g., conjugated to a trigger moiety, therapeuticmoiety and/or other type of moiety), compositions comprising the same,and methods of using the same in the diagnosis, imaging and treatment ofdisease (e.g., cancer, inflammatory disease, chronic pain, etc.).

The present invention provides a multiplicity of linkers and agents(e.g., therapeutic agent, imaging agent, targeting agent, triggeringagent) that find use in the present invention. Indeed, the presentinvention is not limited to any particular linker or to any particulartargeting agent or to any particular dendrimer. In some embodiments, thepresent invention provides a dendrimer conjugated to a linker that isconjugated to an agent (e.g., therapeutic agent, imaging agent,targeting agent, triggering agent), and methods of generating and usingthe same (e.g., to treat cancer, pain and/or inflammation, etc.). Insome embodiments, a dendrimer conjugated to a linker that is conjugatedto an agent (e.g., therapeutic agent, imaging agent, targeting agent,triggering agent) decreases the number of conjugation steps required toform a dendrimer (e.g., a dendrimer conjugate (e.g., a dendrimerconjugated to a targeting agent, imaging agent, therapeutic agent and/ortriggering agent)). For example, in some embodiments, the presentinvention provides a customizable dendrimer wherein one or a pluralityof linkers (e.g. attached to one or a plurality of targeting agents,triggering agents and/or therapeutic agents) are conjugated to adendrimer, thereby decreasing the number of conjugation steps used toform a dendrimer (e.g., versus a dendrimer that is conjugated to atargeting moiety in one step and that is separately conjugated to alinker (e.g., comprising a therapeutic agent, imaging agent, triggeringagent or other moiety) in an additional conjugation step). In someembodiments, a linker conjugated to one or more agents (e.g.,therapeutic agents, imaging agents, targeting agents, triggering agents)is conjugated to one or more additional moieties including, but notlimited to, a therapeutic agent, a triggering agent, an imaging agent, atriggering agent, etc. Thus, in some embodiments, the present inventionprovides a dendrimer with increased load capacity (e.g., increased loadof therapeutic, imaging agent, etc. on the dendrimer). In someembodiments, two or more linkers (e.g., conjugated to one or a pluralityof targeting agents) are conjugated to a dendrimer via the same ordifferent linkage (e.g., covalent linkage).

Several different schemes were evaluated for generating dendrimerconjugates wherein a dendrimer is conjugated to one or more linkers thatcomprise multiple sites for binding (e.g., covalent binding) moieties.For example, in one embodiment, a linker may comprise a chemicalstructure that allows, for example, conjugation of a targeting moietyand a therapeutic compound to the linker. Thus, in some embodiments, adendrimer conjugate of the present invention permits control of thestoichiometry between targeting agent and therapeutic compound (e.g.,generation of one to one ratio, two to one ratio, one to two ratio, oneto three ratio etc. between targeting and therapeutic moieties).

In some embodiments, a dendrimer conjugated to a linker that isconjugated to a targeting agent and/or therapeutic agent comprises alinker that is configured to be irreversibly degraded (e.g., that isnon-reversible (e.g., that permits drug delivery at the correct timeand/or at the correct place)).

In some embodiments, the present invention provides dendrimer molecules(e.g., polyamideamine (PAMAM) dendrimers, polypropylamine (POPAM)dendrimers, or PAMAM-POPAM dendrimers) conjuguated to one or more painrelief agents (e.g., prodrug analgesic molecules). In some embodiments,the dendrimers conjugated to one or more pain relief agents (e.g.,prodrug analgesic molecules) are configured for controlled and/orsustained release of the pain relief agents (e.g., through use oftargeting agents, linking agents, and/or trigger agents conjugated tothe dendrimer and/or pain relief agent). In some embodiments, the painrelief agent conjugated to the dendrimer is active upon administrationto a subject. In some embodiments, sustained release (e.g., slow releaseover a period of 24-48 hours) of the pain relief agent is accomplishedthrough conjugating the pain relief agent to the dendrimer through, forexample, a linkage agent connected to a trigger agent that slowlydegrades in a biological system (e.g., ester linkage). In someembodiments, constitutively active release of the pain relief agent isaccomplished through conjugating the pain relief agent to the dendrimerthrough, for example, a linkage agent connected to a trigger agent thatrenders the pain relief agent constitutively active in a biologicalsystem (e.g., amide linkage, ether linkage). In some embodiments, thedendrimers conjugated to one or more pain relief agents simultaneouslyconfigured for sustained release (e.g., a slow release mechanism thatachieves analgesic concentrations over a period of, for example, 24-48hours) of the pain relief agent prevents adverse side effects of thepain relief agent (e.g., respiratory failure, adverse cardiovascularconsequences). The present invention further provides systems andmethods for treating and/or managing pain through utilization ofdendrimers conjugated to one or more pain relief agents.

In some embodiments, as shown in FIG. 32A, the present inventionprovides compositions comprising a plurality of pain relief agents(e.g., Ketamine and Lorazepam) coupled to dendrimers with a linkageagent connected to a trigger agent that slowly degrades in a biologicalsystem (e.g., amide linkage, ester linkage, ether linkage) (as shown inFIG. 32A, the trigger agent is an ester bond that is released by serumesterases to mediate sustained-release analgesia). When administeredtogether, Ketamine and Lorazepam have favorable analgesic andanxiolytic/amnestic qualities, and relatively broad therapeutic indexes.Such compositions comprising Ketamine and Lorazepam provide analgesiawithout the cardiovascular effects of opioids and minimize their majorcomplication, that being respiratory depression. As a feedbackmechanism, compositions comprising a pain relief agent antagonist (e.g.,Doxapram) complexed with dendrimer through charge interaction that wouldbe released by, for example, acidosis. While acidosis could be observedfrom causes other than respiratory depression, this feedback mechanismis unique in that Doxapram stimulates respirations without reducinganalgesia. As such, there would not be an issue in releasing theDoxapram regardless of the source of the acidosis. Indeed, the increasedrespiratory drive would be of benefit to compensate acidosis even whencaused by metabolic sources from traumatic injuries, hemorrhagic shock,burns or rhabdomyolysis. One advantage to this approach is that itprovides safe analgesia with easily achieved physiological feedback.

For example, in some embodiments, G5 dendrimers with differentpercentages of succinamic acid termini (Scheme 3) wherein Doxaprammolecules are encapsulated in the interior of dendrimers, In suchembodiments, the negative charges on the dendrimer surface prevent therelease of the drug due to the strong electrostatic interaction with thepositively charged Doxapram hydrochloride. In such embodiments, the drugis released once the dendrimer surface carboxyl groups are protonatedwith decreasing pH.

In some embodiments, as shown in FIG. 32B shows two dendrimer conjugatesdesigned for pain management in a subject. One of the dendrimers isconjugated to a morphine drug through a linkage agent connected to atrigger agent (e.g., ester, amide) permitting sustained release. Theother dendrimer is conjugated to a morphine antagonist (e.g., Naloxone)through a linkage agent connected to a trigger agent (e.g., re-doxlinker) permitting release of the morphine antagonist upon detection ofreduced pO2 levels. Each of the dendrimers are targeted for CNS deliverthrough conjugation of targeting agents specific for the CNS (e.g.,transferrin, a synthetic tetanus toxin fragment). Each of the dendrimersare designed for retention within the CNS through conjugation of lockingagents designed to prevent back diffusion of the dendrimer across theBBB (e.g., pyridinium molecule, which when activated be enzymaticreduction, becomes charged and locks the dendrimer in the CNS).

In some embodiments, as shown in FIG. 33, the present invention providesdendrimer conjugates configured to provide effective analgesia (e.g.,narcotic analgesia (e.g., Morphine)) over prolonged time periods. Insome embodiments, the dendrimer conjugates shown in FIG. 33 aretransported to the CNS, and retained in the CNS to provide, for example,constitutive narcotic analgesia. Such embodiments permit the use ofsmaller analgesic doses, while reducing the peripheral adverse effects.In some embodiments, as shown in FIG. 33, the dendrimer conjugatescomprise i) a targeting agent that enables the conjugate to cross theBBB and target neurons, ii) a locking agent (e.g., a re-dox lockingmodule) to prevent the dendrimer conjugate from diffusing back acrossthe BBB, and iii) a pain relief agent (e.g., narcotic analgesic(Morphine)) or pain relief agent antagonist (e.g., Naloxone) coupled bydifferent linking agents and triggering agents. The dendrimer conjugatesare not limited to particular targeting agents. In some embodiments, thetargeting agent for CNS targeting through crossing the BBB istransferrin (see, e.g., Daniels, T. R., et al., Clinical Immunology,2006. 121(2): p. 159-176; Daniels, T. R., et al., Clinical Immunology,2006. 121(2): p. 144-158; each herein incorporated by reference in theirentireties). In some embodiments, the targeting agent for neurontargeting is a 12 amino acid peptide (Tet 1) (see, e.g., Liu, J. K., etal., Neurobiology of Disease, 2005. 19(3): p. 407-418; hereinincorporated by reference in its entirety). The dendrimer conjugates arenot limited to particular locking agents. In some embodiments, thelocking agent for locking the dendrimer conjugate within the CNS is the1,4-dihydrotrigonelline⇄trigonelline (coffearine) re-dox system wherethe lipophilic 1,4-dihydro form (L) is converted in vivo to thehydrophilic quaternary form (L⁺) by oxidation to prevent the dendrimerconjugate from diffusing back into the circulation (see, e.g., Bodor, N.and P. Buchwald, Drug Discovery Today, 2002. 7(14): p. 766-774; hereinincorporated by reference in its entirety). In some embodiments, thedendrimer conjugate device is eliminated from the CNS (e.g., because ofacquired hydrophilicity due to loss of the quaternary form). In someembodiments, the pain relief agent and/or pain relief agent antagonistis attached to the dendrimer through, for example, triggering agentsdesigned for delayed release (e.g., ester bonds, amide bonds, etherbonds). In some embodiments wherein the dendrimer conjugate comprises apain relief agent antagonist (e.g., Naloxone), the pain relief agentantagonist is attached to the dendrimer through a linkage agentconnected to a trigger agent (e.g., re-dox linker) permitting release ofthe pain relief agent antagonist upon detection of reduced pO2 levels.

In some embodiments, the present invention provides a dendrimerconjugate as shown in FIG. 1. For example, FIG. 1 shows a targetingagent (T.A.) conjugated to a linker that is also conjugated to a drug,wherein the linker conjugated to a drug and targeting agent isconjugated to a dendrimer conjugated to an imaging agent (I.A.). In someembodiments, the present invention provides a dendrimer conjugate asshown in FIG. 2 (e.g., possessing targeted anticancer therapeuticmoiety). For example, FIG. 2 shows several structures of dendrimerconjugates, wherein R1, R2, R3 and R4 are each independently selectedfrom hydrogen, halogen, and alkyl. In some embodiments, the alkyl isstraight or cyclic, unsubstituted or substituted (e.g., by from 1 to 4substituents (e.g., selected from the group comprising, but not limitedto, halogen, amino, monoalkylamino, dialkylamino, hydroxy, alkoxy,nitro, aryl, cyano, carboxyl, carboxamide, monoalkylcarboxamide,dialkylcarboxamide, thiol, thioalkyl and sulfonic acid)). In someembodiments, the “U” moiety is present or absent. In some embodiments,when the “U” moiety is absent, one of the R1, R2, R3 and/or R4 groups islinked to a targeting agent through a linker and/or spacer. In someembodiments, R5 is an alkyl (e.g., that is straight chained, branched,cyclic (e.g., that is substituted or unsubtituted)). In someembodiments, R6 is a hydrogen or an alkyl (e.g., of 1-4 carbons (e.g.,that are straight chained or cyclic (e.g., that is substituted orunsubtituted)). In some embodiments, Ra, Rb, Rc, Rd and Re are eachindependently selected from hydrogen, halogen, and alkyl. In someembodiments, the alkyl is straight or cyclic, unsubstituted orsubstituted (e.g., by from 1 to 4 substituents (e.g., selected fromhalogen, amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, nitro,aryl, cyano, carboxyl, carboxamide, monoalkylcarboxamide,dialkylcarboxamide, thiol, thioalkyl and sulfonic acid. In someembodiments, the “U” moiety is present or absent. In some embodiments,when the “U” moiety is absent, one of the Ra, Rb, Rc, Rd and Re groupsis linked to a targeting agent through a linker and/or spacer. In someembodiments, “Y” is an oxygen atom. In some embodiments, “Y” is twohydrogen atoms. In some embodiments, G5 is a generation five poly(amidoamine) (PAMAM) dendrimer (e.g., conjugated to one or more imagingagents (e.g., FITC, etc.), although higher (e.g., G6, G7, G8, G9, G10 orhigher, or lower, G4, G3, or G2 dendrimers may also be used. In someembodiments, “W” is a linker comprising 1-8 carbon and/or nitrogen atoms(e.g., straight chanined, branched, or cyclic, unsubstituted orsubstituted by “R” groups as described above.

In some embodiments, the present invention provides a dendrimerconjugate as shown in FIGS. 3 and 4. In particular, a dendrimerconjugate as shown in FIG. 3 comprises a dendrimer (e.g., a G5 PAMAMdendrimer conjugated to an imaging agent (e.g., FITC) and/or targetingagent) conjugated to a trigger molecule that is conjugated to a linkerthat is conjugated to a therapeutic. A dendrimer conjugate as shown inFIG. 4 comprises a dendrimer (e.g., a G5 PAMAM dendrimer conjugated toan imaging agent (e.g., FITC) and/or targeting agent) conjugated to alinker that is conjugated to a trigger and to a therapeutic moiety. Theconjugates of FIGS. 3 and 4 are configured to be non-toxic to normalcells. For example, the conjugates are configured in such a way so as torelease their therapeutic agent only at a specific, targeted site (e.g.,through activation of a trigger molecule that in to leads to release ofthe therapeutic agent) For example, once a conjugate arrives at a targetsite in a subject (e.g., a tumor, or a site of inflammation), componentsin the target site (e.g., a tumor associated factor, or an inflammatoryor pain associated factor) interacts with the trigger moiety therebyinitiating cleavage of this unit from the linker. In some embodiments,once the trigger is cleaved from the linker (e.g., by a targetassociated moiety, the linker proceeds through spontaneous chemicalbreakdown thereby releasing the therapeutic agent at the target site(e.g., in its active form). The present invention is not limited to anyparticular target associated moiety (e.g., that interacts with andinitiates cleavage of a trigger). In some embodiments, the targetassociated moiety is a tumor associated factor (e.g., an enzyme (e.g.,glucuronidase and/or plasmin), a cathepsin, a matrix metalloproteinase,a hormone receptor (e.g., integrin receptor, hyaluronic acid receptor,luteinizing hormone-releasing hormone receptor, etc.), cancer and/ortumor specific DNA sequence), an inflammatory associated factor (e.g.,chemokine, cytokine, etc.) or other moiety.

Although an understanding of a mechanism of action is not necessary topractice the present invention, and the present invention is not limitedto any particular mechanism of action, in some embodiments, a dendrimerconjugate as described in FIG. 3 or 4 provides a therapeutic to a siteby a mechanism as shown in FIG. 5 or 6. For example, as shown in FIG. 5,a dendrimer conjugate comprising a dendrimer (e.g., a G5 PAMAM dendrimerconjugated to an imaging agent (e.g., FITC) and/or targeting agent)conjugated to a trigger molecule that is conjugated to a linker that isconjugated to a therapeutic (A) interacts with a target associatedmoiety thereby activating the trigger and initiating cleavage of same,releasing the linker therapeutic drug conjugate. Once cleavage of thetrigger occurs, the linker (B) proceeds through a spontaneous chemicalbreakdown at the target site, releasing (e.g., irreversibly releasing)the therapeutic drug at the target site. In some embodiments, as shownin FIG. 6, a dendrimer conjugate comprising a dendrimer (e.g., a G5PAMAM dendrimer conjugated to an imaging agent (e.g., FITC) and/ortargeting agent) conjugated to a linker that is conjugated to a triggerand to a therapeutic moiety (A) interacts with a target associatedmoiety thereby activating the trigger and initiating cleavage of same,releasing a dendrimer-linker-therapeutic moiety from the trigger. Oncecleavage of the trigger occurs, the linker (B) proceeds through aspontaneous chemical breakdown (e.g., to a point where the therapeuticdrug is released from the dendrimer linker conjugate) at the targetsite, releasing (e.g., irreversibly releasing) the therapeutic drug atthe target site. In some embodiments, cleavage of the trigger andsubsequent linker breakdown is not necessary to deliver the therapeuticdrug to the target site. Several design processes for generating adendrimer conjugate comprising a trigger are shown in FIGS. 7 and 8. Thedendrimer conjugates for the present invention (e.g., the dendrimerconjugates as shown in FIGS. 32, 3 and 4) are not limited to anyparticular dendrimer. Indeed, the conjugates may comprise a variety ofdifferent types of dendrimers. In some embodiments, the dendrimer is aPAMAM dendrimer (e.g., G3, G5 or G7 dendrimer). In some embodiments, oneor more amino groups present on the dendrimer are linked (e.g., througha covalent bond) to one or more targeting agents (e.g., folic acid)and/or imaging agents (e.g., FITC) (e.g., as described in U.S. Pat. Nos.6,471,968 and 7,078,461; U.S. Patent Pub. Nos. 20020165179 and20070041934 and WO 06/033766, each of which is hereby incorporated byreference in its entirety for all purposes).

In some embodiments, the present invention provides a dendrimerconjugate as shown in FIG. 9. In particular, a dendrimer conjugate asshown in FIG. 9 comprises a dendrimer (e.g., a G5 PAMAM dendrimerconjugated to an imaging agent (e.g., FITC) and/or targeting agent)conjugated to a trigger molecule that is conjugated to a linker that isconjugated to a therapeutic, or a dendrimer (e.g., a G5 PAMAM dendrimerconjugated to an imaging agent (e.g., FITC) and/or targeting agent)conjugated to a linker that is conjugated to a trigger and to atherapeutic moiety). For example, FIG. 9 shows several structures ofdendrimer conjugates, wherein R1, R2, R3 and R4 are each independentlyselected from hydrogen, halogen, and alkyl. In some embodiments, thealkyl is straight or cyclic, unsubstituted or substituted (e.g., by from1 to 4 substituents (e.g., selected from the group comprising, but notlimited to, halogen, amino, monoalkylamino, dialkylamino, hydroxy,alkoxy, nitro, aryl, cyano, carboxyl, carboxamide, monoalkylcarboxamide,dialkylcarboxamide, thiol, thioalkyl and sulfonic acid. In someembodiments, R5 is an alkyl that is straight, branched or cyclic, thatis unsubstituted or substituted. In some embodiments, R6 is a hydrogenor alkyl of 1-4 carbons that are straight, branched or cyclic, that isunsubstituted or substituted. In some embodiments, the two R6 areconnected together to form a ring of 306 members. In some embodiments,R′, R″, R′″ and R″″ are each independently selected from hydrogen,halogen, and alkyl. In some embodiments, the alkyl is straight orcyclic, unsubstituted or substituted (e.g., by from 1 to 4 substituents(e.g., selected from the group comprising, but not limited to, halogen,amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, nitro, aryl,cyano, carboxyl, carboxamide, monoalkylcarboxamide, dialkylcarboxamide,thiol, thioalkyl and sulfonic acid. In some embodiments, X, X2 and X3are either oxygen or “NR”, wherein “N” is a nitrogen atom, and “R” is analkyl that is straight or branched or cyclic (e.g., substituted orunsubstituted). In some embodiments, “Y” is an oxygen atom or twohydrogen atoms. In some embodiments, A-B is an ethylene group (e.g.,unsubstituted or substituted by alkyls (e.g., straight or cyclic). Insome embodiments, A-B are connected by a carbon chain (e.g., of 2, 3, 4,5, or more carbons) and/or hetero atoms (e.g., forming a saturated orunsaturated aromatic ring structure (e.g., comprising substituents suchas R1, R2, R3 and R4). In some embodiments, G5 is a dendrimer (e.g., aG5 PAMAM dendrimer conjugated to an imaging agent (e.g., FITC) and/ortargeting agent). As described herein, the present invention is notlimited to any particular dendrimer. In some embodiments, “W” is alinker (e.g., comprising a carbon or nitrogen chain (e.g., 2, 3, 4, 5,6, 7, 8, 9, or more carbons or nitrogens (e.g., straight or branched orcyclic (e.g., substituted or unsubstituted (e.g., with R groups asdescribed above))).

The present invention is not limited by the type of dendrimer conjugate(e.g., comprising a trigger) for use in treating a subject. In someembodiments, the dendrimer conjugates of the present invention (see,e.g., FIGS. 32 and 33) are used as delivery agents for pain reliefagents and pain relief agent antagonists. Such dendrimer conjugates arenot limited to uses within particular settings. Indeed, the dendrimerconjugates of the present invention (see, e.g., FIGS. 32 and 33) may beused in any setting requiring treatment and/or management of pain (e.g.,battlefield, ambulance, hospital, clinic, rescue, etc.). In addition,the present invention contemplates dendrimer conjugates comprising oneor more pain relief agent prodrugs and/or pain relief agent antagonistprodrugs developed for site specific conversion to drug based on tumorassociated factors (e.g., hypoxia and pH, tumor-associated enzymes,and/or receptors). In some embodiments, dendrimer conjugates of thepresent invention are configured such that a prodrug (e.g., pain reliefagent prodrug, pain relief agent antagonist prodrug) is conjugated to alinker that is further conjugated to a targeting moiety (e.g., thattargets the conjugate to a particular body region (e.g., CNS)). Althoughan understanding of the mechanism is not necessary for the presentinvention, and the present invention is not limited to any particularmechanism of action, in some embodiments, a trigger component serves asa precursor for site-specific activation. For example, in someembodiments, once the trigger recognizes a particular condition (e.g.,hypoxia), cleavage and/or processing of the trigger is induced, therebyreleasing the pain relief agent and/or pain relief antagonist.

The present invention is not limited to a particular trigger agent or toany particular cleavage and/or processing of the trigger agent. In someembodiments, the present invention provides pain relief agents and/orpain relief agent antagonists coupled to dendrimers with a linkage agentconnected to a trigger agent that slowly degrades in a biological system(e.g., amide linkage, ester linkage, ether linkage) (as shown in FIG.32A, the trigger agent is an ester bond that is released by serumesterases to mediate sustained-release analgesia).

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) hypoxia (e.g., as described in Example 8). Hypoxia is afeature of several disease states, including cancer, inflammation andrheumatoid arthritis, as well as an indicator of respiratory depression(e.g., resulting from analgesic drugs). Advances in the chemistry ofbioreductive drug activation have led to the design of varioushypoxia-selective drug delivery systems in which the pharmacophores ofdrugs are masked by reductively cleaved groups. In some embodiments, adendrimer conjugate of the present invention utilizes a quinone, N-oxideand/or (hetero)aromatic nitro groups. For example, a quinone present ina dendrimer conjugate of the present invention is reduced to phenolunder hypoxia conditions, with spontaneous formation of lactone thatserves as a driving force for drug release (e.g., as shown in FIG. 18).In some embodiments, a heteroaromatic nitro compound present in adendrimer conjugate of the present invention is reduced to either anamine or a hydroxylamine, thereby triggering the spontaneous release ofa therapeutic agent/drug (e.g., as shown in FIG. 19). In someembodiments, the present invention provides pain relief agents and/orpain relief agent antagonists coupled to dendrimers with a linkage agentconnected to a trigger agent that degrades upon detection of reduced pO2concentrations (e.g., through use of a re-dox linker).

The concept of prodrug systems in which the pharmacophores of drugs aremasked by reductively cleavable groups has been widely explored by manyresearch groups and pharmaceutical companies (see, e.g., Beall, H. D.,et al., Journal of Medicinal Chemistry, 1998. 41(24): p. 4755-4766;Ferrer, S., D. P. Naughton, and M. D. Threadgill, Tetrahedron, 2003.59(19): p. 3445-3454; Naylor, M. A., et al., Journal of MedicinalChemistry, 1997. 40(15): p. 2335-2346; Phillips, R. M., et al., Journalof Medicinal Chemistry, 1999. 42(20): p. 4071-4080; Zhang, Z., et al.,Organic & Biomolecular Chemistry, 2005. 3(10): p. 1905-1910; each ofwhich are herein incorporated by reference in their entireties). Severalsuch hypoxia activated prodrugs have been advanced to clinicalinvestigations, and work in relevant oxygen concentrations to preventcerebral damage. The present invention is not limited to particularhypoxia activated trigger agents. In some embodiments, the hypoxiaactivated trigger agents include, but are not limited to, indoquinones,nitroimidazoles, and nitroheterocycles (see, e.g., Damen, E. W. P., etal., Bioorganic & Medicinal Chemistry, 2002. 10(1): p. 71-77; Hay, M.P., et al., Journal of Medicinal Chemistry, 2003. 46(25): p. 5533-5545;Hay, M. P., et al., Journal of the Chemical Society-Perkin Transactions1, 1999(19): p. 2759-2770; each herein incorporated by reference intheir entireties). The mechanism of re-dox triggered release of drugsfrom these linkers is shown in Scheme 2.

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or that associates with a tumor associated enzyme. Insome embodiments, the present invention provides a dendrimer conjugatecomprising a trigger that is sensitive to (e.g., is cleaved by) and/orthat associates with a glucuronidase. Glucuronic acid can be attached toseveral anticancer drugs via various linkers. These anticancer drugsinclude, but are not limited to, doxorubicin, paclitaxel, docetaxel,5-fluorouracil, 9-aminocamtothecin, as well as other drugs underdevelopment. These prodrugs are generally stable at physiological pH andare significantly less toxic than the parent drugs. In some embodiments,dendrimer conjugates comprising anticancer prodrugs find use fortreating necrotic tumors (e.g., that liberate β-glucuronidase) or forADEPT with antibodies that can deliver β-glucuronidase to target tumorcells.

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or that associates with brain enzymes. For example,trigger agents such as indolequinone are reduced by brain enzymes suchas, for example, diaphorase (see, e.g., Damen, E. W. P., et al.,Bioorganic & Medicinal Chemistry, 2002. 10(1): p. 71-77; hereinincorporated by reference in its entirety). For example, in suchembodiments, the antagonist is only active when released during hypoxiato prevent respiratory failure.

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or that associates with a protease. The presentinvention is not limited to any particular protease. In someembodiments, the protease is a cathepsin. In some embodiments, a triggercomprises a Lys-Phe-PABC moiety (e.g., that acts as a trigger). In someembodiments, a Lys-Phe-PABC moiety linked to doxorubicin, mitomycin C,and paclitaxel are utilized as a trigger-therapeutic conjugate in adendrimer conjugate provided herein (e.g., that serve as substrates forlysosomal cathepsin B or other proteases expressed (e.g., overexpressed)in tumor cells. In some embodiments, utilization of a 1,6-eliminationspacer/linker is utilized (e.g., to permit release of therapeutic drugpost activation of trigger).

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or that associates with plasmin. The serine proteaseplasmin is over expressed in many human tumor tissues. Tripeptidespecifiers (e.g., including, but not limited to, Val-Leu-Lys) have beenidentified and linked to anticancer drugs through elimination orcyclization linkers.

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or that associates with a matrix metalloproteases(MMPs). In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) and/or that associates with β-Lactamase (e.g., a β-Lactamaseactivated cephalosporin-based prodrug).

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or activated by a receptor (e.g., expressed on a targetcell (e.g., a tumor cell)). Thus, in some embodiments, a dendrimerconjugate comprises a receptor binding motif conjugated to a therapeuticagent (e.g., cytotoxic drug) thereby providing target specificity.Examples include, but are not limited to, a dendrimer conjugatecomprising a prodrug (e.g., of doxorubicin and/or paclitaxel) targetingintegrin receptor, a hyaluronic acid receptor, and/or a hormone receptor

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger agent that is sensitive to (e.g., iscleaved by) and/or activated by a nucleic acid. Nucleic acid triggeredcatalytic drug release can be utilized in the design of chemotherapeuticagents. Thus, in some embodiments, disease specific nucleic acidsequence is utilized as a drug releasing enzyme-like catalyst (e.g., viacomplex formation with a complimentary catalyst-bearing nucleic acidand/or analog). In some embodiments, the release of a therapeutic agentis facilitated by the therapeutic component being attached to a labileprotecting group, such as, for example, cisplatin or methotrexate beingattached to a photolabile protecting group that becomes released bylaser light directed at cells emitting a color of fluorescence (e.g., inaddition to and/or in place of target activated activation of a triggercomponent of a dendrimer conjugate). In some embodiments, thetherapeutic device also may have a component to monitor the response ofthe tumor to therapy. For example, where a therapeutic agent of thedendrimer induces apoptosis of a target cell (e.g., a cancer cell (e.g.,a prostate cancer cell)), the caspase activity of the cells may be usedto activate a green fluorescence. This allows apoptotic cells to turnorange, (combination of red and green) while residual cells remain red.Any normal cells that are induced to undergo apoptosis in collateraldamage fluoresce green.

In some embodiments, the present invention provides a dendrimerconjugate comprising a linker that connects to a therapeutic compound.In some embodiments, the linker is configured such that itsdecomposition leads to the liberation (e.g., non-reversible liberation)of the therapeutic agent (e.g., pain relief agent) (e.g., at the targetsite (e.g., site of tumor, CNS, and/or inflammatory site)). The linkermay influence multiple characteristics of a dendrimer conjugateincluding, but not limited to, properties of the therapeutic agent(e.g., stability, pharmacokinetic, organ distribution, bioavailability,and/or enzyme recognition (e.g., when the therapeutic agent (e.g.,prodrug)) is enzymatically activated)).

In some embodiments, the linker is an elimination linker. For example,in some embodiments, in a dendrimer conjugate of the present invention,when a trigger is cleaved (e.g., enzymatically and/or chemically), aphenol or an aniline promotes a facile 1,4 or 1,6 elimination, followedby release of a CO₂ molecule and the unmasked therapeutic agent (e.g.,drug) (See, e.g., FIG. 20). In some embodiments, a dendrimer conjugateof the present invention utilizes this configuration and/or strategy tomask one or more hydroxyl groups and/or amino groups of the therapeuticagents. In some embodiments, a linker present within a dendrimerconjugate of the present invention is fine tuned (e.g., to optimizestability and/or drug release from the conjugate). For example, thesizes of the aromatic substituents can be altered (e.g., increased ordecreased) and/or alkyl substitutions at the benzylic position may bemade to alter (e.g., increase or decrease) degradation of the linkerand/or release of the therapeutic agent (e.g., prodrug). In someembodiments, elongated analogs (e.g., double spacers) are used (e.g., todecrease steric hindrance (e.g., for large therapeutic agents (e.g., SeeFIG. 21))). In some embodiments, a dendrimer conjugate of the presentinvention comprises an enol based linker (e.g., that undergoes anelimination reaction to release therapeutic agent (e.g., prodrug)).

In some embodiments, the linker is a cyclization based linker. Forexample, one configuration for this approach is shown in FIG. 22. Anucleophilic group (e.g., OH or NHR) that becomes available once thetrigger is cleaved attacks the carbonyl of the C(O)X— Therapeuticagent/drug (e.g., thereby leading to release of therapeutic agent-XH)and thereby to quickly release the Drug-XH. In some embodiments, adriving force that permits the reaction to reach completion is thestability of the cyclic product. In some embodiments, a cyclizationbased linker of a dendrimer conjugate of the present invention include,but are not limited to, those shown in FIG. 23.

In some embodiments, a dendrimer conjugate of the present inventioncomprises a combination of one or more linkers. For example, in someembodiments, a dendrimer conjugate comprises a combination of two ormore elimination linkers. In some embodiments, a dendrimer conjugate ofthe present invention comprises two or more cyclization linkers. In someembodiments, a dendrimer conjugate of the present invention comprises aone or more elimination linkers and one or more cyclization linkers, ora combination of one or more different types of linkers describedherein. For example, in some embodiments, a dendrimer conjugatecomprises a linker as shown in FIG. 24.

In some embodiments, a dendrimer conjugate of the present inventioncomprises branched self-elimination linkers (e.g., as shown in FIG. 25).Thus, in some embodiments, use of branched linkers provides a conjugatethat can present increased concentrations of a therapeutic agent to atarget site (e.g., inflammatory site, tumor site, etc.).

In some embodiments, a dendrimer conjugate of the present invention isgenerated by a process comprising conjugating a pre-formed tripartitepiece (e.g., trigger, linker, and therapeutic agent) to a dendrimer(e.g., a G5 PAMAM dendrimer or other type of dendrimer described herein(e.g., conjugated to one or more different types of agents (e.g.,imaging agent)). In some embodiments, linkage between a tripartite pieceand a dendrimer comprises a non-cleavable bond (e.g., an ether or anamide bond (e.g., thereby decreasing unwanted activation of a triggerand/or degradation of a linker and/or release of therapeutic drug). Insome embodiments, a linker (e.g., linear or other type of linkerdescribed herein) is utilized to attach a tripartite moiety (e.g.,trigger, linker, and therapeutic agent) to a dendrimer (e.g., in orderto increase drug release, decrease steric hindrance, and/or increasestability of the dendrimer). For example, in some embodiments, thepresent invention provides a dendrimer conjugate as shown in FIG. 26A-B.

In some embodiments, a dendrimer conjugate of the present inventioncomprises a dendrimer conjugated to a linker (e.g., optionallyconjugated to a trigger) that is conjugated to a therapeutic agent. Insome embodiments, the dendrimer conjugate comprises a self-immolativeconnector between an ester bond (e.g., that is to be cleaved) and thetherapeutic agent (e.g., thereby enhancing drug release). For example,although a mechanism is not necessary to practice the present inventionand the present invention is not limited to any particular mechanism ofaction, in some embodiments, a dendrimer conjugate of the presentinvention comprising an ester linkage undergoes esterase catalyzedhydrolysis (e.g., as shown in FIG. 27 (e.g., G5 dendrimer comprising aself-degradable spacer and therapeutic agent)). Thus, in contrast to adendrimer comprising a simple ester (e.g., a dendrimer in the topportion of FIG. 27 wherein therapeutic agent release may or may notoccur, e.g., if x=NH), in some embodiments, the present inventionprovides a dendrimer conjugate comprising an elimination linker (e.g., a1, 6, elimination linker/spacer as shown in the bottom portion of FIG.27 (e.g., that permits complete hydrolysis of the linker (e.g., at atarget site))).

The present invention is not limited by the type of linkerconfiguration. In some embodiments, the linker is conjugated via a freeamino group via an amide linkage (e.g., formed from an active ester(e.g., the N-hydroxysuccinimide ester)). In some embodiments, an esterlinkage remains in the conjugate after conjugation. In some embodiments,linkage occurs through a lysine residue. In some embodiments,conjugation occurs through a short-acting, degradable linkage. Thepresent invention is not limited by the type of degradable linkageutilized. Indeed, a variety of linkages are contemplated to be useful inthe present invention including, but not limited to, physiologicallycleavable linkages including ester, carbonate ester, carbamate, sulfate,phosphate, acyloxyalkyl ether, acetal, and ketal linkages. In someembodiments, a dendrimer conjugate comprises a cleavable linkage presentin the linkage between the dendrimer and linker and/or targeting agentand/or therapeutic agent present therein (e.g., such that when cleaved,no portion of the linkage remains on the dendrimer). In someembodiments, a dendrimer conjugate comprises a cleavable linkage presentin the linker itself (e.g., such that when cleaved, a small portion ofthe linkage remains on the dendrimer).

The present invention is not limited by the type of therapeutic agentdelivered via a dendrimer of the present invention. For example, atherapeutic agent may be any agent selected from the group comprising,but not limited to, a pain relief agent, a pain relief agent antagonist,a chemotherapeutic agent, an anti-oncogenic agent, an anti-angiogenicagent, a tumor suppressor agent, an anti-microbial agent, or anexpression construct comprising a nucleic acid encoding a therapeuticprotein. Illustrative examples of these types of agents are describedherein.

In some embodiments, the therapeutic agent is a pain relief agent. Thedendrimer conjugates of the present invention are not limited to aparticular type or kind of pain relief agent.

In some embodiments, the pain relief agents include several medicationsthat have been used for field deployment and have a proven efficacy formilitary medical applications (see, e.g., Emergency war surgery. 3rd ed.2004, Department of Defense, USA; herein incorporated by reference inits entirety) (see Table 1). These drugs include, but are not limitedto, Ketamine, narcotics (e.g., Morphine, fentanyl, hydromorphone),benzodiazepines (e.g., midazolam, diazepam, Lorazepam) and the selectiveantagonist of narcotics (e.g., Naloxone) and benzodiazepines (e.g.,flumazenil). Military relevance is supported by the fact, for example,that small amounts of Morphine and Ketamine are used by medics duringextraction-evacuation of the injured from the battle field.

TABLE 1 Drug Levels to Target Per 12-Hour Period 12-hour Drug InfusionPer Hour Delivery Coverage Ketamine 1 mg/kg/hr will provide 1 mg × 75 kg=> 75 mg/hr release  900 mg analgesia and anesthesia. Lorazepam 50μg/kg/hr for sedation 50 μg × 75 = 3750 μg/hr or 3.75 mg/hr 45000 μg (45mg) release Morphine 30 μg/kg/hr provides 30 μg × 75 kg is 2250 ug/hrreleased 27000 μg (27 mg) “basal-low end” analgesia Naloxone 5 μg/k/hrprovides basal 5 μg × 75 kg is 375 ug/hr released  9000 μg (9 mg)reversal of narcotic induced side effects Doxapram 2 (to 3) mg/kg/hr 2mg × 75 kg is 150 mg/hr released  1800 mg μg = Micrograms Reference:Micromedex 2006. Assumptions: Requirements for a 75 Kg individual over a12 hours period; delivered in one subcutaneous or intramuscularadministration of maximal volume 5 ml.

In some embodiments, the pain relief agent is Ketamine. Ketamine is apotent analgesic, amnestic and anxiolytic, even in the low dose rangewhile amnesia extends beyond its analgesic duration. Ketamine'stherapeutic index is large and as levels are increased, generalanesthesia is achieved. Unlike other current general anesthetic agents,vital functions (e.g., neuromuscular tone, airway patency, respirations,and cardiovascular function) are maintained. All narcotic agents (e.g.,Morphine) display effects opposite of those of Ketamine with respect tovital functions. As narcotic levels are increased, respirations,neuromuscular tone, and airway patency are decreased whilecardiovascular function may also be compromised, particularly due toperipheral vasodilatation. Ketamine also induces bronchodilation, whichis particularly useful when irritants cause bronchoconstriction andcoughing. Morphine suppresses coughing and either leaves bronchomotortone unaltered or increases it (see, e.g., Anesthesia. 4th ed. 1994,Churchill Livingstone: New York; Goodman & Gilman's the pharmacologicalbasis of therapeutics. 9th ed. 1996, McGraw-Hill, Health ProfessionsDivision: New York; each herein incorporated by reference in itsentirety). When faced with severe injuries and blood loss, low doseKetamine provides analgesia and amnesia while preserving homeostaticmechanisms and vital functions. Ketamine levels can be increased toachieve a state of “dissociation” in which major procedures (e.g., anamputation) can be accomplished with cardio respiratory stability whilethe individual seems unattached to the procedure. Dissociation is uniqueto Ketamine.

In some embodiments, the present invention provides dendrimer conjugatescomprising Ketamine and Lorazepam. Unfortunately, disforic reactions canoccur in a small percentage of recipients, but can be effectivelytreated with the concurrent administration of benzodiazepines (e.g.,Lorazepam). Lorazepam has excellent amnestic and anxiolytic properties,which are very desirable in the severely injured combatant (see, e.g.,Anesthesia. 4th ed. 1994, Churchill Livingstone: New York; Goodman &Gilman's the pharmacological basis of therapeutics. 9th ed. 1996,McGraw-Hill, Health Professions Division: New York; each hereinincorporated by reference in its entirety). It does not have analgesicnor anesthetic properties. It has mild, centrally mediated musclerelaxant properties while it is an anticonvulsant. Its effects onhomeostasis of the respiratory and hemodynamic system are mildlydepressant when used in the dose range of its anxiolytic properties.

In some embodiments, the pain relief agent is Morphine. Morphine is thestandard against which all other analgesics are compared. It is lesspotent as an analgesic when compared to Ketamine. Its sedation can beaccompanied by euphoria, but its amnestic and anxiolytic effects areless when compared to Ketamine and Lorazepam. Even in high doses,Morphine is a poor anesthetic and an unreliable amnestic, but theseproperties may maintain co-operativity on the battlefield. Morphine'sanalgesic effects overlap closely with its effects on homeostasis of therespiratory and hemodynamic system. Thus as the dosage of Morphine isincreased, depression of respirations, and loss of airway patency andreflexes become soon apparent relative to Ketamine's effect. Itshemodynamic effects include veno-vasodilatation within the range ofMorphine's analgesia. Thus, in the severely injured with blood loss,Morphine's analgesic range is limited by its effects on homeostasis ofvital functions. Morphine's therapeutic index, particularly in thesetting of the severely injured combatant, is low compared to the indexfor Ketamine. Thus, Morphine has many good qualities but should beadministered in a manner to avoid side effects.

In some embodiments, the pain relief agent antagonist is Doxapram.Doxapram is a respiratory stimulant causing an increase in tidal volumewith an increase in respiratory rate used in acute respiratoryinsufficiency. It can improve cardiac output in the setting ofhypovolemia. It may increase catecholamines release. Doxapram is usefulas a respiratory and cardiovascular stimulant in the battlefield fieldsetting to reduce or negate respiratory and hemodynamic effects of anyproposed analgesic-amnestic-anxiolytic agents. Thus, the release ofDoxapram is a viable counter-regulatory effect for respiratorydepression whatever the cause.

In some embodiments, the pain relief agent antagonist is Naloxone.Naloxone is an effective, selective opioid (e.g., Morphine) antagonist.It reverses a range of Morphine's effects including Morphine's analgesiaand respiratory depression. Although Morphine's analgesic andrespiratory ranges overlap, low dose infusions of Naloxone can reverseMorphine's respiratory depression while its analgesic effect isrelatively unaffected and pain-relief can remain present. Thus, it is aprime candidate for a dendrimer-drug delivery system requiring aMorphine feedback mechanism.

In some embodiments, pain relief agents include, but are not limited to,analgesic drugs and respective antagonists. Examples of analgesic drugsinclude, but are not limited to, paracetamol and Non-steroidalanti-inflammatory drugs (NSAIDs), COX-2 inhibitors, opiates andmorphonimimetics, and specific analgesic agents.

Examples of NSAIDs include, but are not limited to, salicylates (e.g.,Acetylsalicylic acid (Aspirin), Amoxiprin, Benorylate/Benorilate,Choline magnesium salicylate, Diflunisal, Ethenzamide, Faislamine,Methyl salicylate, Magnesium salicylate, Salicyl salicylate,Salicylamide), arylalkanoic acids (e.g., Diclofenac, Aceclofenac,Acemethacin, Alclofenac, Bromfenac, Etodolac, Indometacin, Nabumetone,Oxametacin, Proglumetacin, Sulindac, Tolmetin), 2-arylpropionic acids(profens) (e.g., Ibuprofen, Alminoprofen, Benoxaprofen, Carprofen,Dexibuprofen, Dexketoprofen, Fenbufen, Fenoprofen, Flunoxaprofen,Flurbiprofen, Ibuproxam, Indoprofen, Ketoprofen, Ketorolac, Loxoprofen,Naproxen, Oxaprozin, Pirprofen, Suprofen, Tiaprofenic acid),N-arylanthranilic acids (fenamic acids) (e.g., Mefenamic acid,Flufenamic acid, Meclofenamic acid, Tolfenamic acid), pyrazolidinederivatives (e.g., Phenylbutazone, Ampyrone, Azapropazone, Clofezone,Kebuzone, Metamizole, Mofebutazone, Oxyphenbutazone, Phenazone,Sulfinpyrazone), oxicams (e.g., Piroxicam, Droxicam, Lornoxicam,Meloxicam, Tenoxicam), sulphonanilides (e.g., nimesulide), licofelone,and omega-3 fatty acids.

Examples of COX-2 inhibitors include, but are not limited to Celecoxib,Etoricoxib, Lumiracoxib, Parecoxib, Rofecoxib, Valdecoxib.

Examples of Opiates include, but are not limited to, natural opiates(e.g., alkaloids contained in the resin of the opium poppy includingmorphine, codeine and thebaine), semi-synthetic opiates (e.g., createdfrom the natural opioids, such as hydromorphone, hydrocodone, oxycodone,oxymorphone, desomorphine, diacetylmorphine (Heroin), nicomorphine,dipropanoylmorphine, diamorphine, benzylmorphine, Buprenorphine,Nalbuphine, Pentazocine, meperidine, diamorphine, and ethylmorphine),fully synthetic opioids (e.g., such as fentanyl, pethidine, Oxycodone,Oxymorphone, methadone, tramadol, Butorphanol, Levorphanol, andpropoxyphene), and endogenous opioid peptides (e.g., produced naturallyin the body, such as endorphins, enkephalins, dynorphins, andendomorphins).

Additional analgesics include, but are not limited to, tricyclicantidepressants (e.g., amitriptyline, carbamazepine, gabapentin, andpregabalin), Tetrahydrocannabinol, ketamine, clonidine,α₂-adrenoreceptor agonists, mexiletine, Orphenadrine, cyclobenzaprine,scopolamine, atropine, gabapentin, first-generation antidepressants andother drugs possessing anticholinergic and/or antispasmodic.

In some embodiments, pain relief agents include anesthetic drugs andrespective antagonists. Examples of anesthetic drugs include, but arenot limited to, local anesthetics (e.g., procaine, amethocaine, cocaine,lidocaine, prilocaine, bupivacaine, levobupivacaine, ropivacaine,dibucaine), inhaled anesthetics (e.g., Desflurane, Enflurane, Halothane,Isoflurane, Nitrous oxide, Sevoflurane, Xenon), intravenous anesthetics(e.g., Barbiturates (e.g., amobarbital (Amytal), pentobarbital(Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital,Thiopental, Methylphenobarbital, Metharbital, Barbexaclone)),Benzodiazepines (e.g., alprazolam, bromazepam (Lexotan),chlordiazepoxide (Librium), Clobazam, Clonazepam, Clorazepate, Diazepam,Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam,Flunitrazepam, oxazepam (Serax), temazepam (Restoril, Normison, Planum,Tenox, and Temaze), Triazolam), Etomidate, Ketamine, Propofol).

In some embodiments, pain relief agents include anticonvulsant drugs andrespective antagonists. Examples of anticonvulsant drugs include, butare not limited to, aldehydes (e.g., paraldehyde), aromatic allylicalcohols (e.g., stiripentol), barbiturates (e.g., amobarbital (Amytal),pentobarbital (Nembutal), secobarbital (Seconal), Phenobarbital,Methohexital, Thiopental, Methylphenobarbital, Metharbital,Barbexaclone), benzodiazepines (e.g., alprazolam, bromazepam (Lexotan),chlordiazepoxide (Librium), Clobazam, Clonazepam, Clorazepate, Diazepam,Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam,Flunitrazepam, oxazepam (Serax), temazepam (Restoril, Normison, Planum,Tenox, and Temaze), Triazolam), bromides (e.g., potassium bromide),carbamates (e.g., felbamate), carboxamides (e.g., carbamazepine,oxcarbazepine), fatty acids (e.g., valproates (e.g., valproic acid,sodium valproate, and divalproex sodium), Vigabatrin, Progabide,Tiagabine), fructose derivatives (e.g., topiramate), gaba analogs (e.g.,gabapentin, pregabalin), hydantoins (e.g., Ethotoin, Phenytoin,Mephenytoin, Fosphenytoin), Oxazolidinediones (e.g., paramethadione,trimethadione, ethadione), priopionates (e.g., primidone), pyrrolidines(e.g., brivaracetam, levetiracetam, seletracetam), succinimides (e.g.,Ethosuximide, Phensuximide, Mesuximide), sulfonamides (e.g.,Acetazolamide, Sulthiame, Methazolamide, Zonisamide), triazines (e.g.,lamotrigine), ureas (e.g., pheneturide, phenacemide), and valproylamdies(amide derivatives of valproate) (e.g., valpromide, valnoctamide).

In some embodiments, pain relief agents include mood stablizer drugs.Examples of mood stabilizer drugs include, but are not limited to,Lithium carbonate, lithium orotate, lithium salt, Valproic acid(Depakene), divalproex sodium (Depakote), sodium valproate (Depacon),Lamotrigine (Lamictal), Carbamazepine (Tegretol), Gabapentin(Neurontin), Oxcarbazepine (Trileptal), and Topiramate (Topamax).

In some embodiments, pain relief agents include psycholeptic drugs.Examples of psycholeptic drugs include, but are not limited to,anxiolytic drugs, antipsychotic drugs, and hypnotic drugs, and sedativedrugs. Examples of anxiolytic drugs include, but are not limited to,benzodiazepines (e.g., alprazolam, bromazepam (Lexotan),chlordiazepoxide (Librium), Clobazam, Clonazepam, Clorazepate, Diazepam,Midazolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam,Flunitrazepam, oxazepam (Serax), temazepam (Restoril, Normison, Planum,Tenox, and Temaze), Triazolam), serotonin 1A agonists (e.g., Buspirone(BuSpar)), barbituates (e.g., amobarbital (Amytal), pentobarbital(Nembutal), secobarbital (Seconal), Phenobarbital, Methohexital,Thiopental, Methylphenobarbital, Metharbital, Barbexaclone),hydroxyzine, cannabidiol, and herbal treatments. (e.g., valerian, kava(Kava Kava), chamomile, Kratom, Blue Lotus extracts, Sceletium tortuosum(kanna) and bacopa monniera). are reputed to have anxiolytic properties.Examples of antipsychotic drugs include, but are not limited to,butyrophenones (e.g., haloperidol), phenothiazines (e.g., Chlorpromazine(Thorazine), Fluphenazine (Prolixin), Perphenazine (Trilafon),Prochlorperazine (Compazine), Thioridazine (Mellaril), Trifluoperazine(Stelazine), Mesoridazine, Promazine, Triflupromazine (Vesprin),Levomepromazine (Nozinan), Promethazine (Phenergan)), thioxanthenes(e.g., Chlorprothixene, Flupenthixol (Depixol and Fluanxol), Thiothixene(Navane), Zuclopenthixol (Clopixol & Acuphase)), clozapine, olanzapine,Risperidone (Risperdal), Quetiapine (Seroquel), Ziprasidone (Geodon),Amisulpride (Solian), Paliperidone (Invega), dopamine, bifeprunox,norclozapine (ACP-104), Aripiprazole (Abilify), Tetrabenazine, andCannabidiol. Examples of hypnotics include, but are not limited to,Barbiturates, Opioids, benzodiazepines (e.g., alprazolam, bromazepam(Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam,Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam,nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam),nonbenzodiazepines (e.g., Zolpidem, Zaleplon, Zopiclone, Eszopiclone),antihistamines (e.g., Diphenhydramine, Doxylamine, Hydroxyzine,Promethazine), gamma-hydroxybutyric acid (Xyrem), Glutethimide, Chloralhydrate, Ethchlorvynol, Levomepromazine, Chlormethiazole, Melatonin, andAlcohol. Examples of sedatives include, but are not limited to,barbituates (e.g., amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone), benzodiazepines (e.g.,alprazolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam,Clonazepam, Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam,temazepam, nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax),temazepam (Restoril, Normison, Planum, Tenox, and Temaze), Triazolam),Herbal sedatives (e.g., ashwagandha, catnip, kava (Piper methysticum),mandrake, marijuana, valerian), solvent sedatives (e.g., chloral hydrate(Noctec), diethyl ether (Ether), ethyl alcohol (alcoholic beverage),methyl trichloride (Chloroform)), nonbenzodiazepine sedatives (e.g.,eszopiclone (Lunesta), zaleplon (Sonata), zolpidem (Ambien), zopiclone(Imovane, Zimovane)), clomethiazole (clomethiazole),gamma-hydroxybutyrate (GHB), Thalidomide, ethchlorvynol (Placidyl),glutethimide (Doriden), ketamine (Ketalar, Ketaset), methaqualone(Sopor, Quaalude), methyprylon (Noludar), and ramelteon (Rozerem).

In some embodiments, pain relief agents include psychoanaleptic drugs.Examples of psychoanaleptic drugs include, but are not limited to,antidepressants, psychostimulants, and anti-dementia drugs. Examples ofantidepresants include, but are not limited to, selective serotoninreuptake inhibitors (SSRIs) (e.g., fluoxetine (Prozac), paroxetine(Paxil, Seroxat), escitalopram (Lexapro, Esipram), citalopram (Celexa),and sertraline (Zoloft)), serotonin-norepinephrine reuptake inhibitors(SNRIs) (e.g., venlafaxine (Effexor), and duloxetine (Cymbalta)),noradrenergic and specific serotonergic antidepressants (NASSAs) (e.g.,mirtazapine (Avanza, Zispin, Remeron)), norepinephrine (noradrenaline)reuptake inhibitors (NRIs) (e.g., reboxetine (Edronax)),norepinephrine-dopamine reuptake inhibitors (e.g., bupropion(Wellbutrin, Zyban)), tricyclic antidepressants (TCAs) (e.g.,amitriptyline and desipramine), monoamine oxidase inhibitor (MAOIs)(e.g., phenelzine (Nardil), moclobemide (Manerix), selegiline), andaugmentor drugs (e.g., tryptophan (Tryptan) and buspirone (Buspar)).Examples of psychostimulants include, but are not limited to,amphetamine, methamphetamine, cocaine, methylphenidate, and arecoline).Examples of anti-dementia drugs include, but are not limited to,Acetylcholinesterase inhibitors (e.g., Tacrine (Cognex), donepezil(Aricept), galantamine (Razadyne), and rivastigmine (Exelon).

In some embodiments, pain relief agents include muscle relaxant drugs.Examples of muscle relaxant drugs include, but are not limited to,depolarizing muscle relaxants (e.g., Succinylcholine), short actingnon-depolarizing muscle relaxants (e.g., Mivacurium, Rapacuronium),intermediate acting non-depolarizing muscle relaxants (e.g., Atracurium,Cisatracurium, Rocuronium, Vecuronium), and long acting non-depolarizingmuscle relaxants (e.g., Alcuronium, Doxacurium, Gallamine, Metocurine,Pancuronium, Pipecuronium, d-Tubocurarine).

In some embodiments, the pain relief agent antagonists include drugsthat counter the effect (e.g., side effect, main effect, cardiovasculareffect) of a pain relief agent. The present invention is not limited toparticular pain relief agent antagonists (e.g., Anesthetic antagonists,Analgesic antagonists, Anticonvulsant antagonists, Mood stabilizerantagonists, Psycholeptic drug antagonists, Psychoanaleptic drugantagonists, and muscle relaxant antagonists). In some embodiments, thepain relief agent antagonists include, but are not limited to,respiratory stimulants (e.g., Doxapram, BIMU-8, CX-546), opiod receptorantagonists (e.g., Naloxone, naltrexone, nalorphine, levallorphan,cyprodime, naltrindole, norbinaltorphimine, buprenorphine), agents thateffect of benzodiazepines (e.g., flumazenil), agents that reverse theeffect of non-depolarizing muscle relaxants (e.g., neostigmine).

In some embodiments, a dendrimer conjugated comprising a linker maycomprise nearly any therapeutic agent (e.g., pain relief agent, painrelief agent antagonist) comprising a hydroxyl and/or amino group. Insome embodiments, the therapeutic agent is an anti-cancer drug or agent.For example, in some embodiments, the therapeutic agent is doxorubicin(or an analog thereof) or paclitaxel (or an analog thereof). In someembodiments, a dendrimer conjugate of the invention comprises atherapeutic agent comprising a single reactive group (e.g., at a primaryor secondary position). In some embodiments, a dendrimer conjugate ofthe present invention is synthesized utilizing a selectiveprotection/deprotection strategy if multiple functional groups arepresent within a therapeutic agent. In some embodiments, a dendrimerconjugate of the present invention provides the ability to deliver atherapeutic agent that, when not in the context of the dendrimerconjugate (e.g., in the absence of conjugation to a dendrimer (e.g., adendrimer comprising a linker and a trigger (e.g., configured to shieldand/or mask the therapeutic drug and/or prohibit release of thetherapeutic drug until the dendrimer reaches and reacts with a targetsite))) is toxic to a subject (e.g., that is too toxic to be utilized totreat a subject). Thus, in some embodiments, the present inventionprovides dendrimer conjugates comprising therapeutic agents that sufferfrom delivery issues and/or toxicity issues and/or non-specificityissues in the absence of being conjugated to a dendrimer conjugate. Forexample, in some embodiments, the present invention provides a dendrimerconjugate comprising a therapeutic agent comprising a compound of thecamptothecin family (e.g., IRINOTECAN). IRINOTECAN is a prodrug of10-hydroxycamptothecin (SN-38), which is 1000-fold more cytotoxic thanIRINOTECAN. It has been reported that the conversion of irinotecan tohydroxycamptothecin has very low efficiency. Thus, in some embodiments,the present invention provides a dendrimer conjugate comprisinghydroxycamptothecin (See, e.g., FIG. 28).

In some embodiments of the present invention, the therapeutic agentincludes, but is not limited to, a chemotherapeutic agent, ananti-oncogenic agent, an anti-angiogenic agent, a tumor suppressoragent, an anti-microbial agent, or an expression construct comprising anucleic acid encoding a therapeutic protein, although the presentinvention is not limited by the nature of the therapeutic agent. Infurther embodiments, the therapeutic agent is protected with aprotecting group selected from photo-labile, radio-labile, andenzyme-labile protecting groups. In some embodiments, thechemotherapeutic agent is selected from a group consisting of, but notlimited to, platinum complex, verapamil, podophylltoxin, carboplatin,procarbazine, mechloroethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, adriamycin,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, bleomycin, etoposide, tamoxifen, paclitaxel, taxol,transplatinum, 5-fluorouracil, vincristin, vinblastin, bisphosphonate(e.g., CB3717), chemotherapeutic agents with high affinity for folicacid receptors, ALIMTA (Eli Lilly), and methotrexate. In someembodiments, the anti-oncogenic agent comprises an antisense nucleicacid (e.g., RNA, molecule). In certain embodiments, the antisensenucleic acid comprises a sequence complementary to an RNA of anoncogene. In preferred embodiments, the oncogene includes, but is notlimited to, ab1, Bc1-2, Bc1-xL, erb, fms, gsp, hst, jun, myc, neu, raf;ras, ret, src, or trk. In some embodiments, the nucleic acid encoding atherapeutic protein encodes a factor including, but not limited to, atumor suppressor, cytokine, receptor, inducer of apoptosis, ordifferentiating agent. In preferred embodiments, the tumor suppressorincludes, but is not limited to, BRCA1, BRCA2, C-CAM, p16, p21, p53,p73, Rb, and p27. In preferred embodiments, the cytokine includes, butis not limited to, GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, β-interferon,γ-interferon, and TNF. In preferred embodiments, the receptor includes,but is not limited to, CFTR, EGFR, estrogen receptor, IL-2 receptor, andVEGFR. In preferred embodiments, the inducer of apoptosis includes, butis not limited to, AdE1B, Bad, Bak, Bax, Bid, Bik, Bim, Harakid, andICE-CED3 protease. In some embodiments, the therapeutic agent comprisesa short-half life radioisotope.

In some embodiments of the present invention, the biological monitoringagent comprises an agent that measures an effect of a therapeutic agent(e.g., directly or indirectly measures a cellular factor or reactioninduced by a therapeutic agent), however, the present invention is notlimited by the nature of the biological monitoring agent. In someembodiments, the monitoring agent is capable of detecting (e.g.,measuring) apoptosis caused by the therapeutic agent.

In some embodiments of the present invention, the imaging agentcomprises a radioactive label including, but not limited to ¹⁴C, ³⁶Cl,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, 125I, ¹³¹I, ¹¹¹Ln, ¹⁵²Eu, ⁵⁹Fe, ⁶⁷Ga, ³²P, , 186-Re,³⁵S, ⁷⁵Se, Tc-99m, and ¹⁷⁵Yb. In some embodiments, the imaging agentcomprises a fluorescing entity. In a preferred embodiment, the imagingagent is fluorescein isothiocyanate or 6-TAMARA.

Dendrimer conjugates of the present invention are not limited by thetype of anti-angiogenic agent used. Indeed, a variety of anti-angiogenicagents are contemplated to be useful in the compositions of the presentinvention including, but not limited to, Batimastat, Marimastat, AG3340,Neovastat, PEX, TIMP-1, -2, -3, -4, PAI-1, -2, uPA Ab, uPAR Ab,Amiloride, Minocycline, tetracyclines, steroids, cartilage-derived TIMP,αvβ3 Ab: LM609 and Vitaxin, RGD containing peptides, αvβ5 Ab,Endostatin, Angiostatin, aaAT, IFN-α, IFN-γ, IL-12, nitric oxidesynthase inhibitors, TSP-1, TNP-470, Combretastatin A4, Thalidomide,Linomide, IFN-α, PF-4, prolactin fragment, Suramin and analogues, PPS,distamycin A analogues, FGF-2 Ab, antisense-FGF-2, Protamine, SU5416,soluble Flt-1, dominant-negative Flk-1, VEGF receptor ribosymes, VEGFAb, Aspirin, NS-398, 6-AT, 6A5BU, 7-DX, Genistein, Lavendustin A, Ang-2,batimastat, marimastat, anti-αvβ3 monoclonal antibody (LM609)thrombospondin-1 (TSP-1) Angiostatin, endostatin, TNP-470,Combretastatin A-4, Anti-VEGF antibodies, soluble Flk-1, Flt-1receptors, inhibitors of tyrosine kinase receptors, SU5416,heparin-binding growth factors, pentosan polysulfate, platelet-derivedendothelial cell growth factor/Thymidine phosphorylase (PD-ECGF/TP), cox(e.g., cox-1 an cox-2) inhibitors (e.g., Celebrex and Vioxx), DT385,Tissue inhibitor of metalloprotease (TIMP-1, TIMP-2), Zinc, Plasminogenactivator-inhibitor-1 (PAI-1), p53 Rb, Interleukin-10 Interleukin-12,Angiopoietin-2, Angiotensin, Angiotensin II (AT2 receptor), Caveolin-1,caveolin-2, Angiopoietin-2, Angiotensin, Angiotensin II (AT2 receptor),Caveolin-1, caveolin-2, Endostatin, Interferon-alpha, Isoflavones,Platelet factor-4, Prolactin (16 Kd fragment), Thrombospondin,Troponin-1, Bay 12-9566, AG3340, CGS 27023A, CGS 27023A, COL-3,(Neovastat), BMS-275291, Penicillamine, TNP-470 (fumagillin derivative),Squalamine, Combretastatin, Endostatin, Penicillamine, FarnesylTransferase Inhibitor (FTI), -L-778,123, -SCH66336, -R115777, anti-VEGFantibody, Thalidomide, SU5416, Ribozyme, Angiozyme, SU6668,PTK787/ZK22584, Interferon-alpha, Interferon-alpha, Suramin, Vitaxin,EMD121974, Penicillamine, Tetrathiomolybdate, Captopril, serine proteaseinhibitors, CAI, ABT-627, CM101/ZDO101, Interleukin-12, IM862,PNU-145156E, those described in U.S. Patent App. No. 20050123605, hereinincorporated by reference in its entirety, and fragments or portions ofthe above that retain anti-angiogenic (e.g., angiostatic or inhibitoryproperties).

The present invention is not limited to any particular targeting agent.In some embodiments, targeting agents are conjugated to the dendrimersfor delivery of the dendrimers to desired body regions (e.g., to thecentral nervous system (CNS). The targeting agents are not limited totargeting specific body regions. In some embodiments, the targetingagents target the central nervous system (CNS). In some embodiments,where the targeting agent is specific for the CNS, the targeting agentis transferrin (see, e.g., Daniels, T. R., et al., Clinical Immunology,2006. 121(2): p. 159-176; Daniels, T. R., et al., Clinical Immunology,2006. 121(2): p. 144-158; each herein incorporated by reference in theirentireties). Transferrin has been utilized as a targeting vector totransport, for example, drugs, liposomes and proteins across the BBB byreceptor mediated transcytosis (see, e.g., Smith, M. W. and M.Gumbleton, Journal of Drug Targeting, 2006. 14(4): p. 191-214; hereinincorporated by reference in its entirety). In some embodiments, thetargeting agents target neurons within the central nervous system (CNS).In some embodiments, where the targeting agent is specific for neuronswithin the CNS, the targeting agent is a synthetic tetanus toxinfragment (e.g., a 12 amino acid peptide (Tet 1) (HLNILSTLWKYR)) (see,e.g., Liu, J. K., et al., Neurobiology of Disease, 2005. 19(3): p.407-418; herein incorporated by reference in its entirety).

In some embodiments, the targeting agent is a moiety that has affinityfor a tumor associated factor. For example, a number of targeting agentsare contemplated to be useful in the present invention including, butnot limited to, RGD sequences, low-density lipoprotein sequences, aNAALADase inhibitor, epidermal growth factor, and other agents that bindwith specificity to a target cell (e.g., a cancer cell)). In someembodiments, the targeting agent is an antibody, receptor ligand,hormone, vitamin, or antigen. However, the present invention is notlimited by the nature of the targeting agent. In some embodiments, theantibody is specific for a disease-specific antigen. In someembodiments, the disease-specific antigen comprises a tumor-specificantigen. In some embodiments, the receptor ligand includes, but is notlimited to, a ligand for CFTR, EGFR, estrogen receptor, FGR2, folatereceptor, IL-2 receptor, glycoprotein, or VEGFR. In some embodiments,the receptor ligand is folic acid.

The present invention is not limited to cancer and/or tumor targetingagents. Indeed, dendrimers of the present invention can be targeted(e.g., via a linker conjugated to the dendrimer wherein the linkercomprises a targeting agent) to a variety of target cells or tissues(e.g., to a biologically relevant environment) via conjugation to anappropriate targeting agent. For example, in some embodiments, thetargeting agent is a moiety that has affinity for an inflammatory factor(e.g., a cytokine or a cytokine receptor moiety (e.g., TNF-α receptor)).In some embodiments, the targeting agent is a sugar, peptide, antibodyor antibody fragment, hormone, hormone receptor, or the like.

In some embodiments of the present invention, the targeting agentincludes, but is not limited to an antibody, receptor ligand, hormone,vitamin, and antigen, however, the present invention is not limited bythe nature of the targeting agent. In some embodiments, the antibody isspecific for a disease-specific antigen. In some embodiments, thedisease-specific antigen comprises a tumor-specific antigen. In someembodiments, the receptor ligand includes, but is not limited to, aligand for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2receptor, glycoprotein, and VEGFR. In some embodiments, the receptorligand is folic acid.

The present invention also provides a method of treating a medicalcondition and/or a disease (e.g., cancer, inflammatory disease, chronicpain, autoimmune disease, etc.) comprising administering to a subjectsuffering from or susceptible to medical condition and/or a disease atherapeutically effective amount of a composition comprising a dendrimerconjugate (e.g., comprising a linker and/or trigger and a therapeuticagent) described herein. In some embodiments, the medical conditionand/or disease is pain (e.g., chronic pain, mild pain, recurring pain,severe pain, etc.). In some embodiments, the dendrimer conjugates areconfigured to deliver pain relief agents to a subject. In someembodiments, the dendrimer conjugates are configured to deliver painrelief agents and pain relief agent antagonists to counter the sideeffects of pain relief agents. The dendrimer conjugates are not limitedto treating a particular type of pain and/or pain resulting from adisease. Examples include, but are not limited to, pain resulting fromtrauma (e.g., trauma experienced on a battlefield, trauma experienced inan accident (e.g., car accident)).

In some embodiments, the disease is cancer. In some embodiments, thedendrimers are designed for retention within the CNS through conjugationof locking agents designed to prevent back diffusion of the dendrimeracross the BBB (e.g., pyridinium molecule, which when activated byenzymatic reduction, becomes charged and locks the dendrimer in theCNS). The present invention is not limited by the type of cancer treatedusing the compositions and methods of the present invention. Indeed, avariety of cancer can be treated including, but not limited to, cancerslocated within the CNS, prostate cancer, colon cancer, breast cancer,lung cancer and epithelial cancer. Similarly, the present invention isnot limited by the type of inflammatory disease and/or chronic paintreated using the compositions of the present invention. Indeed, avariety of diseases can be treated including, but not limited to,arthritis (e.g., osteoarthritis, rheumatoid arthritis, etc.),inflammatory bowel disease (e.g., colitis, Crohn's disease, etc.),autoimmune disease (e.g., lupus erythematosus, multiple sclerosis,etc.), inflammatory pelvic disease, etc.

In preferred embodiments, dendrimer conjugates of the present inventionare configured such that they are readily cleared from the subject(e.g., so that there is little to no detectable toxicity at efficaciousdoses). In some embodiments, the disease is a neoplastic disease,selected from, but not limited to, leukemia, acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic, (granulocytic) leukemia, chroniclymphocytic leukemia, Polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's disease, Multiple myeloma, Waldenstrom'smacroglobulinemia, Heavy chain disease, solid tumors, sarcomas andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, and neuroblastomaretinoblastoma. In some embodiments, thedisease is an inflammatory disease selected from the group consistingof, but not limited to, eczema, inflammatory bowel disease, rheumatoidarthritis, asthma, psoriasis, ischemia/reperfusion injury, ulcerativecolitis and acute respiratory distress syndrome. In some embodiments,the disease is a viral disease selected from the group consisting of,but not limited to, viral disease caused by hepatitis B, hepatitis C,rotavirus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), human T-cell lymphotropic virustype I (HTLV-I), human T-cell lymphotropic virus type II (HTLV-II),AIDS, DNA viruses such as hepatitis type B and hepatitis type C virus;parvoviruses, such as adeno-associated virus and cytomegalovirus;papovaviruses such as papilloma virus, polyoma viruses, and SV40;adenoviruses; herpes viruses such as herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), and Epstein-Barr virus; poxviruses,such as variola (smallpox) and vaccinia virus; and RNA viruses, such ashuman immunodeficiency virus type I (HIV-I), human immunodeficiencyvirus type II (HIV-II), human T-cell lymphotropic virus type I (HTLV-I),human T-cell lymphotropic virus type II (HTLV-II), influenza virus,measles virus, rabies virus, Sendai virus, picornaviruses such aspoliomyelitis virus, coxsackieviruses, rhinoviruses, reoviruses,togaviruses such as rubella virus (German measles) and Semliki forestvirus, arboviruses, and hepatitis type A virus.

The dendrimers of the present invention find use in the detection andtreatment of a variety of cancers. Indeed, the present invention is notlimited by the type of cancer to be treated. Thus, in some embodiments,the present invention provides compositions comprising dendrimerconjugates for the targeting and identification of angiogenesisassociated with cancers (e.g., carcinomas). For example, in someembodiments, a dendrimer conjugate of the present invention furthercomprises a targeting agent (e.g., folic acid moiety) that associateswith high affinity to a targeting agent ligand (e.g., receptor) on acancer cell (e.g., carcinoma cells and/or solid tumor cells). In someembodiments, dendrimer conjugate and a targeting agent, that target andidentify cancer cells and/or angiogenesis associated with cancer,further comprise a therapeutic agent that inhibits angiogenesis therebytreating the cancer. In some embodiments, treatment with dendrimerconjugates and an anti-angiogenic agent are used in combination withother dendrimers of the present invention, with other chemotherapeutictreatments, and/or as a treatment following surgical removal of a tumoror cancerous tissue. In some embodiments, a targeting moiety (e.g.,folic acid or other targeting moiety described herein) possesses a highaffinity for ligands (e.g., receptors or other types of proteins ormolecules) present on cancer cell possessing such ligands therebypermitting the targeting, identification and treatment of disease (e.g.,cancer) with little to no toxicity to surrounding healthy cells andtissue.

In some embodiments, the present invention also provides a kitcomprising a composition comprising dendrimer conjugate comprising alinker and/or trigger and a therapeutic agent. In some embodiments, thekit comprises a fluorescent agent or bioluminescent agent.

Some embodiments of the present invention provide compositionscomprising dendrimer conjugates further comprising one or morefunctional groups, the functional groups including, but not limited to,therapeutic agents, biological monitoring components, biological imagingcomponents, targeting components, and components to identify thespecific signature of cellular abnormalities. As such, in someembodiments, a therapeutic dendrimer conjugate of the present inventionis made up of individual dendrimers, each with one or more functionalgroups being specifically conjugated with or covalently linked to thedendrimer.

As is clear from the above example, the use of the compositions of thepresent invention facilitates non-intrusive sensing, signaling, andintervention for treating and managing pain, cancer and other diseasesand conditions. Since specific protocols of molecular alterations incancer cells are identified using this technique, non-intrusive sensingthrough the dendrimers is achieved and may then be employedautomatically against various tumor phenotypes. The present invention isnot limited to a particular type of dendrimer.

Indeed, dendrimeric polymers have been described extensively (See, e.g.,Tomalia, Advanced Materials 6:529 (1994); Angew, Chem. Int. Ed. Engl.,29:138 (1990); incorporated herein by reference in their entireties).Dendrimer polymers are synthesized as defined spherical structurestypically ranging from 1 to 20 nanometers in diameter. Methods formanufacturing a G5 PAMAM dendrimer with a protected core is shown (FIGS.1-5). In preferred embodiments, the protected core diamine isNH2-CH2-CH2-NHPG. Molecular weight and the number of terminal groupsincrease exponentially as a function of generation (the number oflayers) of the polymer (See, e.g., FIG. 9). Different types ofdendrimers can be synthesized based on the core structure that initiatesthe polymerization process (See e.g., FIGS. 1-5).

The dendrimer core structures dictate several characteristics of themolecule such as the overall shape, density and surface functionality(See, e.g., Tomalia et al., Chem. Int. Ed. Engl., 29:5305 (1990)).Spherical dendrimers can have ammonia as a trivalent initiator core orethylenediamine (EDA) as a tetravalent initiator core (See, e.g., FIG.9). Recently described rod-shaped dendrimers (See, e.g., Yin et al., J.Am. Chem. Soc., 120:2678 (1998)) use polyethyleneimine linear cores ofvarying lengths; the longer the core, the longer the rod. Dendriticmacromolecules are available commercially in kilogram quantities and areproduced under current good manufacturing processes (GMP) forbiotechnology applications.

Dendrimers may be characterized by a number of techniques including, butnot limited to, electrospray-ionization mass spectroscopy, ¹³C nuclearmagnetic resonance spectroscopy, ¹H nuclear magnetic resonancespectroscopy (See, e.g., Example 5, FIG. 10(A) and Example 7, FIG. 14),high performance liquid chromatography (See, e.g., Example 5, FIG.10(B); and Example 6, FIG. 13), size exclusion chromatography withmulti-angle laser light scattering (See, e.g., Example 4, FIG. 8),ultraviolet spectrophotometry (See, e.g., Example 8, FIG. 17), capillaryelectrophoresis and gel electrophoresis. These tests assure theuniformity of the polymer population and are important for monitoringquality control of dendrimer manufacture for GMP applications and invivo usage.

Numerous U.S. Patents describe methods and compositions for producingdendrimers. Examples of some of these patents are given below in orderto provide a description of some dendrimer compositions that may beuseful in the present invention, however it should be understood thatthese are merely illustrative examples and numerous other similardendrimer compositions could be used in the present invention.

U.S. Pat. No. 4,507,466, U.S. Pat. No. 4,558,120, U.S. Pat. No.4,568,737, and U.S. Pat. No. 4,587,329 each describe methods of makingdense star polymers with terminal densities greater than conventionalstar polymers. These polymers have greater/more uniform reactivity thanconventional star polymers, i.e. 3rd generation dense star polymers.These patents further describe the nature of the amidoamine dendrimersand the 3-dimensional molecular diameter of the dendrimers.

U.S. Pat. No. 4,631,337 describes hydrolytically stable polymers. U.S.Pat. No. 4,694,064 describes rod-shaped dendrimers. U.S. Pat. No.4,713,975 describes dense star polymers and their use to characterizesurfaces of viruses, bacteria and proteins including enzymes. Bridgeddense star polymers are described in U.S. Pat. No. 4,737,550. U.S. Pat.No. 4,857,599 and U.S. Pat. No. 4,871,779 describe dense star polymerson immobilized cores useful as ion-exchange resins, chelation resins andmethods of making such polymers.

U.S. Pat. No. 5,338,532 is directed to starburst conjugates ofdendrimer(s) in association with at least one unit of carriedagricultural, pharmaceutical or other material. This patent describesthe use of dendrimers to provide means of delivery of highconcentrations of carried materials per unit polymer, controlleddelivery, targeted delivery and/or multiple species such as e.g., drugsantibiotics, general and specific toxins, metal ions, radionuclides,signal generators, antibodies, interleukins, hormones, interferons,viruses, viral fragments, pesticides, and antimicrobials.

U.S. Pat. No. 6,471,968 describes a dendrimer complex comprisingcovalently linked first and second dendrimers, with the first dendrimercomprising a first agent and the second dendrimer comprising a secondagent, wherein the first dendrimer is different from the seconddendrimer, and where the first agent is different than the second agent.

Other useful dendrimer type compositions are described in U.S. Pat. No.5,387,617, U.S. Pat. No. 5,393,797, and U.S. Pat. No. 5,393,795 in whichdense star polymers are modified by capping with a hydrophobic groupcapable of providing a hydrophobic outer shell. U.S. Pat. No. 5,527,524discloses the use of amino terminated dendrimers in antibody conjugates.

The use of dendrimers as metal ion carriers is described in U.S. Pat.No. 5,560,929. U.S. Pat. No. 5,773,527 discloses non-crosslinkedpolybranched polymers having a comb-burst configuration and methods ofmaking the same. U.S. Pat. No. 5,631,329 describes a process to producepolybranched polymer of high molecular weight by forming a first set ofbranched polymers protected from branching; grafting to a core;deprotecting first set branched polymer, then forming a second set ofbranched polymers protected from branching and grafting to the corehaving the first set of branched polymers, etc.

U.S. Pat. No. 5,902,863 describes dendrimer networks containinglipophilic organosilicone and hydrophilic polyanicloamine nanscopicdomains. The networks are prepared from copolydendrimer precursorshaving PAMAM (hydrophilic) or polyproyleneimine interiors andorganosilicon outer layers. These dendrimers have a controllable size,shape and spatial distribution. They are hydrophobic dendrimers with anorganosilicon outer layer that can be used for specialty membrane,protective coating, composites containing organic organometallic orinorganic additives, skin patch delivery, absorbants, chromatographypersonal care products and agricultural products.

U.S. Pat. No. 5,795,582 describes the use of dendrimers as adjuvants forinfluenza antigen. Use of the dendrimers produces antibody titer levelswith reduced antigen dose. U.S. Pat. No. 5,898,005 and U.S. Pat. No.5,861,319 describe specific immunobinding assays for determiningconcentration of an analyte. U.S. Pat. No. 5,661,025 provides details ofa self-assembling polynucleotide delivery system comprising dendrimerpolycation to aid in delivery of nucleotides to target site. This patentprovides methods of introducing a polynucleotide into a eukaryotic cellin vitro comprising contacting the cell with a composition comprising apolynucleotide and a dendrimer polyeation non-covalently coupled to thepolynucleotide.

Dendrimer-antibody conjugates for use in in vitro diagnosticapplications have previously been demonstrated (See, e.g., Singh et al.,Clin. Chem., 40:1845 (1994)), for the production ofdendrimer-chelant-antibody constructs, and for the development ofboronated dendrimer-antibody conjugates (for neutron capture therapy);each of these latter compounds may be used as a cancer therapeutic (See,e.g., Wu et al., Bioorg. Med. Chem. Lett., 4:449 (1994); Wiener et al.,Magn. Reson. Med. 31:1 (1994); Barth et al., Bioconjugate Chem. 5:58(1994); and Barth et al.).

Some of these conjugates have also been employed in the magneticresonance imaging of tumors (See, e.g., Wu et al., (1994) and Wiener etal., (1994), supra). Results from this work have documented that, whenadministered in vivo, antibodies can direct dendrimer-associatedtherapeutic agents to antigen-bearing tumors. Dendrimers also have beenshown to specifically enter cells and carry either chemotherapeuticagents or genetic therapeutics. In particular, studies show thatcisplatin encapsulated in dendrimer polymers has increased efficacy andis less toxic than cisplatin delivered by other means (See, e.g., Duncanand Malik, Control Rel. Bioact. Mater. 23:105 (1996)).

Dendrimers have also been conjugated to fluorochromes or molecularbeacons and shown to enter cells. They can then be detected within thecell in a manner compatible with sensing apparatus for evaluation ofphysiologic changes within cells (See, e.g., Baker et al., Anal. Chem.69:990 (1997)). Finally, dendrimers have been constructed asdifferentiated block copolymers where the outer portions of the moleculemay be digested with either enzyme or light-induced catalysis (See,e.g., Urdea and Hom, Science 261:534 (1993)). This allows the controlleddegradation of the polymer to release therapeutics at the disease siteand provides a mechanism for an external trigger to release thetherapeutic agents.

In some embodiments, dendrimer conjugates of the present inventioncontain one or more signature identifying agents that are activated by,or are able to interact with, a signature component (“signature”). Inpreferred embodiments, the signature identifying agent is an antibody,preferably a monoclonal antibody, that specifically binds the signature(e.g., cell surface molecule specific to a cell to be targeted).

In some embodiments of the present invention, tumor cells areidentified. Tumor cells have a wide variety of signatures, including thedefined expression of cancer-specific antigens such as Muc1, HER-2 andmutated p53 in breast cancer. These act as specific signatures for thecancer, being present in 30% (HER-2) to 70% (mutated p53) of breastcancers. In some embodiments, a dendrimer of the present inventioncomprises a monoclonal antibody that specifically binds to a mutatedversion of p53 that is present in breast cancer. In some embodiments, adendrimer of the present invention comprises an antibody (e.g.,monoclonal antibody) with high affinity for a signature including, butnot limited to, Muc1 and HER-2.

In some embodiments of the present invention, cancer cells expressingsusceptibility genes are identified. For example, in some embodiments,there are two breast cancer susceptibility genes that are used asspecific signatures for breast cancer: BRCA1 on chromosome 17 and BRCA2on chromosome 13. When an individual carries a mutation in either BRCA1or BRCA2, they are at an increased risk of being diagnosed with breastor ovarian cancer at some point in their lives. These genes participatein repairing radiation-induced breaks in double-stranded DNA. It isthought that mutations in BRCA1 or BRCA2 might disable this mechanism,leading to more errors in DNA replication and ultimately to cancerousgrowth.

In addition, the expression of a number of different cell surfacereceptors find use as targets for the binding and uptake of a dendrimerconjugate. Such receptors include, but are not limited to, EGF receptor,folate receptor, FGR receptor 2, and the like.

In some embodiments of the present invention, changes in gene expressionassociated with chromosomal abborations are the signature component. Forexample, Burkitt lymphoma results from chromosome translocations thatinvolve the Myc gene. A chromosome translocation means that a chromosomeis broken, which allows it to associate with parts of other chromosomes.The classic chromosome translocation in Burkitt lymophoma involveschromosome 8, the site of the Myc gene. This changes the pattern of Mycexpression, thereby disrupting its usual function in controlling cellgrowth and proliferation.

From the discussion above it is clear that there are many differenttumor signatures that find use with the present invention, some of whichare specific to a particular type of cancer and others which arepromiscuous in their origin. The present invention is not limited to anyparticular tumor signature or any other disease-specific signature. Forexample, tumor suppressors that find use as signatures in the presentinvention include, but are not limited to, p53, Mud, CEA, p16, p21, p27,CCAM, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-1, MEN-II, p73, VHL, FCC andMCC.

In some embodiments of the present invention, a dendrimer conjugatecomprises at least one imaging agent that can be readily imaged. Thepresent invention is not limited by the nature of the imaging componentused. In some embodiments of the present invention, imaging modulescomprise surface modifications of quantum dots (See e.g., Chan and Nie,Science 281:2016 (1998)) such as zinc sulfide-capped cadmium selenidecoupled to biomolecules (Sooklal, Adv. Mater., 10:1083 (1998)).

In some embodiments, the imaging module comprises dendrimers producedaccording to the “nanocomposite” concept (See, e.g., Balogh et al.,Proc. of ACS PMSE 77:118 (1997) and Balogh and Tomalia, J. Am. Che.Soc., 120:7355 (1998)). In these embodiments, dendrimers are produced byreactive encapsulation, where a reactant is preorganized by thedendrimer template and is then subsequently immobilized in/on thepolymer molecule by a second reactant. Size, shape, size distributionand surface functionality of these nanoparticles are determined andcontrolled by the dendritic macromolecules. These materials have thesolubility and compatibility of the host and have the optical orphysiological properties of the guest molecule (i.e., the molecule thatpermits imaging). While the dendrimer host may vary according to themedium, it is possible to load the dendrimer hosts with differentcompounds and at various guest concentration levels. Complexes andcomposites may involve the use of a variety of metals or other inorganicmaterials. The high electron density of these materials considerablysimplifies the imaging by electron microscopy and related scatteringtechniques. In addition, properties of inorganic atoms introduce new andmeasurable properties for imaging in either the presence or absence ofinterfering biological materials. In some embodiments of the presentinvention, encapsulation of gold, silver, cobalt, iron atoms/moleculesand/or organic dye molecules such as fluorescein are encapsulated intodendrimers for use as nanoscopi composite labels/tracers, although anymaterial that facilitates imaging or detection may be employed. In apreferred embodiment, the imaging agent is fluorescein isothiocyanate

In some embodiments of the present invention, imaging is based on thepassive or active observation of local differences in density ofselected physical properties of the investigated complex matter. Thesedifferences may be due to a different shape (e.g., mass density detectedby atomic force microscopy), altered composition (e.g. radiopaquesdetected by X-ray), distinct light emission (e.g., fluorochromesdetected by spectrophotometry), different diffraction (e.g.,electron-beam detected by TEM), contrasted absorption (e.g., lightdetected by optical methods), or special radiation emission (e.g.,isotope methods), etc. Thus, quality and sensitivity of imaging dependon the property observed and on the technique used. The imagingtechniques for cancerous cells have to provide sufficient levels ofsensitivity to is observe small, local concentrations of selected cells.The earliest identification of cancer signatures requires highselectivity (i.e., highly specific recognition provided by appropriatetargeting) and the highest possible sensitivity.

In some embodiments, once a targeted dendrimer conjugate has attached to(or been internalized into) a target cell (e.g., tumor cell and orinflammatory cell), one or more modules on the device serve to image itslocation. Dendrimers have already been employed as biomedical imagingagents, perhaps most notably for magnetic resonance imaging (MRI)contrast enhancement agents (See e.g., Wiener et al., Mag. Reson. Med.31:1 (1994); an example using PAMAM dendrimers). These agents aretypically constructed by conjugating chelated paramagnetic ions, such asGd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA), towater-soluble dendrimers. Other paramagnetic ions that may be useful inthis context include, but are not limited to, gadolinium, manganese,copper, chromium, iron, cobalt, erbium, nickel, europium, technetium,indium, samarium, dysprosium, ruthenium, ytterbium, yttrium, and holmiumions and combinations thereof. In some embodiments of the presentinvention, a dendrimer conjugate is also conjugated to a targetinggroup, such as epidermal growth factor (EGF), to make the conjugatespecifically bind to the desired cell type (e.g., in the case of EGF,EGFR-expressing tumor cells). In a preferred embodiment of the presentinvention, DTPA is attached to dendrimers via the isothiocyanate of DTPAas described by Wiener (Wiener et al., Mag. Reson. Med. 31:1 (1994)).

Dendrimeric MRI agents are particularly effective due to thepolyvalency, size and architecture of dendrimers, which results inmolecules with large proton relaxation enhancements, high molecularrelaxivity, and a high effective concentration of paramagnetic ions atthe target site. Dendrimeric gadolinium contrast agents have even beenused to differentiate between benign and malignant breast tumors usingdynamic MRI, based on how the vasculature for the latter type of tumorimages more densely (Adam et al., Ivest. Rad. 31:26 (1996)). Thus, MRIprovides a particularly useful imaging system of the present invention.

Static structural microscopic imaging of cancerous cells and tissues hastraditionally been performed outside of the patient. Classical histologyof tissue biopsies provides a fine illustrative example, and has provena powerful adjunct to cancer diagnosis and treatment. After removal, aspecimen is sliced thin (e.g., less than 40 microns), stained, fixed,and examined by a pathologist. If images are obtained, they are mostoften 2-D transmission bright-field projection images. Specialized dyesare employed to provide selective contrast, which is almost absent fromthe unstained tissue, and to also provide for the identification ofaberrant cellular constituents. Quantifying sub-cellular structuralfeatures by using computer-assisted analysis, such as in nuclear ploidydetermination, is often confounded by the loss of histologic contextowing to the thinness of the specimen and the overall lack of 3-Dinformation. Despite the limitations of the static imaging approach, ithas been invaluable to allow for the identification of neoplasia inbiopsied tissue. Furthermore, its use is often the crucial factor in thedecision to perform invasive and risky combinations of chemotherapy,surgical procedures, and radiation treatments, which are oftenaccompanied by severe collateral tissue damage, complications, and evenpatient death.

A dendrimer conjugate of the present invention allows functionalmicroscopic imaging of tumors and provide improved methods for imaging.The methods find use in vivo, in vitro, and ex vivo. For example, in oneembodiment of the present invention, dendrimer conjugates of the presentinvention are designed to emit light or other detectable signals uponexposure to light. Although the labeled dendrimers may be physicallysmaller than the optical resolution limit of the microscopy technique,they become self-luminous objects when excited and are readilyobservable and measurable using optical techniques. In some embodimentsof the present invention, sensing fluorescent biosensors in a microscopeinvolves the use of tunable excitation and emission filters andmultiwavelength sources (See, e.g., Farkas et al., SPEI 2678:200(1997)). In embodiments where the imaging agents are present in deepertissue, longer wavelengths in the Near-infrared (NMR) are used (Seee.g., Lester et al., Cell Mol. Biol. 44:29 (1998)). Dendrimericbiosensing in the Near-IR has been demonstrated with dendrimericbiosensing antenna-like architectures (See, e.g., Shortreed et al., J.Phys. Chem., 101:6318 (1997)). Biosensors that find use with the presentinvention include, but are not limited to, fluorescent dyes andmolecular beacons.

In some embodiments of the present invention, in vivo imaging isaccomplished using functional imaging techniques. Functional imaging isa complementary and potentially more powerful techniques as compared tostatic structural imaging. Functional imaging is best known for itsapplication at the macroscopic scale, with examples including functionalMagnetic Resonance Imaging (fMRI) and Positron Emission Tomography(PET). However, functional microscopic imaging may also be conducted andfind use in in vivo and ex vivo analysis of living tissue. Functionalmicroscopic imaging is an efficient combination of 3-D imaging, 3-Dspatial multispectral volumetric assignment, and temporal sampling: inshort a type of 3-D spectral microscopic movie loop. Interestingly,cells and tissues autofluoresce. When excited by several wavelengths,providing much of the basic 3-D structure needed to characterize severalcellular components (e.g., the nucleus) without specific labeling.Oblique light illumination is also useful to collect structuralinformation and is used routinely. As opposed to structural spectralmicroimaging, functional spectral microimaging may be used withbiosensors, which act to localize physiologic signals within the cell ortissue. For example, in some embodiments of the present invention,biosensor-comprising dendrimers of the present invention are used toimage upregulated receptor families such as the folate or EGF classes.In such embodiments, functional biosensing therefore involves thedetection of physiological abnormalities relevant to carcinogenesis ormalignancy, even at early stages. A number of physiological conditionsmay be imaged using the compositions and methods of the presentinvention including, but not limited to, detection of nanoscopicdendrimeric biosensors for pH, oxygen concentration, Ca²+ concentration,and other physiologically relevant analytes.

In some embodiments, the present invention provides dendrimer conjugateshaving a biological monitoring component. The biological monitoring orsensing component of a dendrimer conjugate of the present invention isone that can monitor the particular response in a target cell (e.g.,tumor cell) induced by an agent (e.g., a therapeutic agent provided bythe therapeutic component of the dendrimer conjugate). While the presentinvention is not limited to any particular monitoring system, theinvention is illustrated by methods and compositions for monitoringcancer treatments. In preferred embodiments of the present invention,the agent induces apoptosis in cells and monitoring involves thedetection of apoptosis. In particular embodiments, the monitoringcomponent is an agent that fluoresces at a particular wavelength whenapoptosis occurs. For example, in a preferred embodiment, caspaseactivity activates green fluorescence in the monitoring component.Apoptotic cancer cells, which have turned red as a result of beingtargeted by a particular signature with a red label, turn orange whileresidual cancer cells remain red. Normal cells induced to undergoapoptosis (e.g., through collateral damage), if present, will fluorescegreen.

In these embodiments, fluorescent groups such as fluorescein areemployed in the monitoring component. Fluorescein is easily attached tothe dendrimer surface via the isothiocyanate derivatives, available fromMOLECULAR PROBES, Inc. This allows the dendrimer conjugate to be imagedwith the cells via confocal microscopy. Sensing of the effectiveness ofthe dendrimer conjugates is preferably achieved by using fluorogenicpeptide enzyme substrates. For example, apoptosis caused by thetherapeutic agents results in the production of the peptidase caspase-1(ICE). CALBIOCHEM sells a number of peptide substrates for this enzymethat release a fluorescent moiety. A particularly useful peptide for usein the present invention is:

(SEQ ID NO: 1) MCA-Tyr-Glu-Val-Asp-Gly-Trp-Lys-(DNP)-NH₂where MCA is the (7-methoxycoumarin-4-yl)acetyl and DNP is the2,4-dinitrophenyl group (See, e.g., Talanian et al., J. Biol. Chem.,272: 9677 (1997)). In this peptide, the MCA group has greatly attenuatedfluorescence, due to fluorogenic resonance energy transfer (FRET) to theDNP group. When the enzyme cleaves the peptide between the aspartic acidand glycine residues, the MCA and DNP are separated, and the MCA groupstrongly fluoresces green (excitation maximum at 325 nm and emissionmaximum at 392 nm).

In some embodiments of the present invention, the lysine end of thepeptide is linked to the dendrimer conjugate, so that the MCA group isreleased into the cytosol when it is cleaved. The lysine end of thepeptide is a useful synthetic handle for conjugation because, forexample, it can react with the activated ester group of a bifunctionallinker such as Mal-PEG-OSu. Thus the appearance of green fluorescence inthe target cells produced using these methods provides a clearindication that apoptosis has begun (if the cell already has a red colorfrom the presence of aggregated quantum dots, the cell turns orange fromthe combined colors).

Additional fluorescent dyes that find use with the present inventioninclude, but are not limited to, acridine orange, reported as sensitiveto DNA changes in apoptotic cells (Abrams et al., Development 117:29(1993)) and cis-parinaric acid, sensitive to the lipid peroxidation thataccompanies apoptosis (Hockenbery et al., Cell 75:241 (1993)). It shouldbe noted that the peptide and the fluorescent dyes are merely exemplary.It is contemplated that any peptide that effectively acts as a substratefor a caspase produced as a result of apoptosis finds use with thepresent invention.

In some embodiments, conjugation between a dendrimer (e.g., terminal armof a dendrimer) and a functional group or between functional groups isaccomplished through use of a 1,3-dipolar cycloaddition reaction (“clickchemistry”). ‘Click chemistry’ involves, for example, the coupling oftwo different moieties (e.g., a therapeutic agent and a functionalgroup) (e.g., a first functional group and a second functional group)via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (orequivalent thereof) on the surface of the first moeity and an azidemoiety (e.g., present on a triazine composition) (or equivalent thereof)(or any active end group such as, for example, a primary amine endgroup, a hydroxyl end group, a carboxylic acid end group, a thiol endgroup, etc.) on the second moiety (see, e.g., U.S. Provisional PatentApp. No. 61/140,480, herein incorporated by reference in its entirety.‘Click’ chemistry is an attractive coupling method because, for example,it can be performed with a wide variety of solvent conditions includingaqueous environments. For example, the stable triazole ring that resultsfrom coupling the alkyne with the azide is frequently achieved atquantitative yields and is considered to be biologically inert (see,e.g., Rostovtsev, V. V.; et al., Angewandte Chemie-International Edition2002, 41, (14), 2596; Wu, P.; et al., Angewandte Chemie-InternationalEdition 2004, 43, (30), 3928-3932; each herein incorporated by referencein their entireties).

In some embodiments, conjugation between a dendrimer (e.g., a terminalarm of a dendrimer) and a functional ligand is accomplished during a“one-pot” reaction. The term “one-pot synthesis reaction” or equivalentsthereof, e.g., “1-pot”, “one pot”, etc., refers to a chemical synthesismethod in which all reactants are present in a single vessel. Reactantsmay be added simultaneously or sequentially, with no limitation as tothe duration of time elapsing between introduction of sequentially addedreactants. In some embodiments, a one-pot reaction occurs wherein ahydroxyl-terminated dendrimer (e.g., HO-PAMAM dendrimer) is reacted withone or more functional ligands (e.g., a therapeutic agent, a pro-drug, atrigger agent, a targeting agent, an imaging agent) in one vessel, suchconjugation being facilitated by ester coupling agents (e.g.,2-chloro-1-methylpyridinium iodide and 4-(dimethylamino)pyridine) (see,e.g., U.S. Provisional Patent App. No. 61/226,993, herein incorporatedby reference in its entirety).

Functionalized nanoparticles (e.g., dendrimers) often contain moieties(including but not limited to ligands, functional ligands, conjugates,therapeutic agents, targeting agents, imaging agents, fluorophores) thatare conjugated to the periphery. Such moieties may for example beconjugated to one or more dendrimer branch termini. Classical multi-stepconjugation strategies used during the synthesis of functionalizeddendrimers generate a stochastic distribution of products with differingnumbers of ligands attached per dendrimer molecule, thereby creating apopulation of dendrimers with a wide distribution in the numbers ofligands attached. The low structural uniformity of such dendrimerpopulations negatively affects properties such as therapeutic potency,pharmacokinetics, or effectiveness for multivalent targeting.Difficulties in quantifying and resolving such populations to yieldsamples with sufficient structural uniformity can pose challenges.However, in some embodiments, use of separation methods (e.g., reversephase chromatography) customized for optimal separation of dendrimerpopulations in conjunction with peak fitting analysis methods allowsisolation and identification of subpopulations of functionalizeddendrimers with high structural uniformity (see, e.g., U.S. ProvisionalPat. App. No. 61/237,172; herein incorporated by reference in itsentirety). In certain embodiments, such methods and systems provide adendrimer product made by the process comprising: a) conjugation of atleast one ligand type to a dendrimer to yield a population ofligand-conjugated dendrimers; b) separation of the population ofligand-conjugated dendrimers with reverse phase HPLC to result insubpopulations of ligand-conjugated dendrimers indicated by achromatographic trace; and c) application of peak fitting analysis tothe chromatographic trace to identify subpopulations ofligand-conjugated dendrimers wherein the structural uniformity of ligandconjugates per molecule of dendrimer within said subpopulation is, e.g.,approximately 80% or more.

As described above, another component of the present invention is thatthe dendrimer conjugate compositions are able to specifically target aparticular tissue region and/or cell type (e.g., CNS). In someembodiments, the dendrimer conjugate targets the CNS (e.g., viatransferrin), neurons within the CNS (e.g., via Teti), the peripheralnervous system, muscles, and/or nerves.

In some embodiments of the present invention, targeting groups areconjugated to dendrimers and/or linkers conjugated to the dendrimerswith either short (e.g., direct coupling), medium (e.g. usingsmall-molecule bifunctional linkers such as SPDP, sold by PIERCECHEMICAL Company), or long (e.g., PEG bifunctional linkers, sold byNEKTAR, Inc.) linkages. Since dendrimers have surfaces with a largenumber of functional groups, more than one targeting group and/or linkermay be attached to each dendrimer. As a result, multiple binding eventsmay occur between the dendrimer conjugate and the target cell. In theseembodiments, the dendrimer conjugates have a very high affinity fortheir target cells via this “cooperative binding” or polyvalentinteraction effect.

For steric reasons, in some embodiments, the smaller the ligands, themore can be attached to the surface of a dendrimer and/or linkersattached thereto. Recently, Wiener reported that dendrimers withattached folic acid would specifically accumulate on the surface andwithin tumor cells expressing the high-affinity folate receptor (hFR)(See, e.g., Wiener et al., Invest. Radiol., 32:748 (1997)). The hFRreceptor is expressed or upregulated on epithelial tumors, includingbreast cancers. Control cells lacking hFR showed no significantaccumulation of folate-derivatized dendrimers. Folic acid can beattached to full generation PAMAM dendrimers via a carbodiimide couplingreaction. Folic acid is a good targeting candidate for the dendrimers,with its small size and a simple conjugation procedure.

Antibodies can be generated to allow for the targeting of antigens orimmunogens (e.g., tumor, tissue or pathogen specific antigens) onvarious biological targets (e.g., pathogens, tumor cells, normaltissue). Such antibodies include, but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

In some embodiments, the antibodies recognize tumor specific epitopes(e.g., TAG-72 (See, e.g., Kjeldsen et al., Cancer Res. 48:2214-2220(1988); U.S. Pat. Nos. 5,892,020; 5,892,019; and 5,512,443); humancarcinoma antigen (See, e.g., U.S. Pat. Nos. 5,693,763; 5,545,530; and5,808,005); TP1 and TP3 antigens from osteocarcinoma cells (See, e.g.,U.S. Pat. No. 5,855,866); Thomsen-Friedenreich (TF) antigen fromadenocarcinoma cells (See, e.g., U.S. Pat. No. 5,110,911); “KC-4antigen” from human prostrate adenocarcinoma (See, e.g., U.S. Pat. Nos.4,708,930 and 4,743,543); a human colorectal cancer antigen (See, e.g.,U.S. Pat. No. 4,921,789); CA125 antigen from cystadenocarcinoma (See,e.g., U.S. Pat. No. 4,921,790); DF3 antigen from human breast carcinoma(See, e.g., U.S. Pat. Nos. 4,963,484 and 5,053,489); a human breasttumor antigen (See, e.g., U.S. Pat. No. 4,939,240); p97 antigen of humanmelanoma (See, e.g., U.S. Pat. No. 4,918,164); carcinoma ororosomucoid-related antigen (CORA)(See, e.g., U.S. Pat. No. 4,914,021);a human pulmonary carcinoma antigen that reacts with human squamous celllung carcinoma but not with human small cell lung carcinoma (See, e.g.,U.S. Pat. No. 4,892,935); T and Tn haptens in glycoproteins of humanbreast carcinoma (See, e.g., Springer et al., Carbohydr. Res.178:271-292 (1988)), MSA breast carcinoma glycoprotein termed (See,e.g., Tjandra et al., Br. J. Surg. 75:811-817 (1988)); MFGM breastcarcinoma antigen (See, e.g., Ishida et al., Tumor Biol. 10:12-22(1989)); DU-PAN-2 pancreatic carcinoma antigen (See, e.g., Lan et al.,Cancer Res. 45:305-310 (1985)); CA125 ovarian carcinoma antigen (See,e.g., Hanisch et al., Carbohydr. Res. 178:29-47 (1988)); YH206 lungcarcinoma antigen (See, e.g., Hinoda et al., (1988) Cancer J. 42:653-658(1988)). Each of the foregoing references are specifically incorporatedherein by reference.

Various procedures known in the art are used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals can be immunized by injection with the peptide corresponding tothe desired epitope including but not limited to rabbits, mice, rats,sheep, goats, etc. In a preferred embodiment, the peptide is conjugatedto an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants are usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Kohler and Milstein (Kohler and Milstein, Nature256:495-497 (1975)), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al. Immunol. Today 4:72(1983)), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 (1985)).

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilizing recent technology (See e.g.,PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., Proc.Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77-96 (1985)).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies.An additional embodiment of the invention utilizes the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,Science 246:1275-1281 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragment thatcan be produced by pepsin digestion of the antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and the Fab fragments that can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art (e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.).

The dendrimer conjugates of the present invention have many advantagesover liposomes, such as their greater stability, better control of theirsize and polydispersity, and generally lower toxicity and immunogenicity(See e.g., Duncan et al, Polymer Preprints 39:180 (1998)). Thus, in someembodiments of the present invention, anti-HER2 antibody fragments, aswell as other targeting antibodies are conjugated to dendrimers, astargeting agents for the nanodevices of the present invention.

The bifunctional linkers SPDP and SMCC and the longer Mal-PEG-OSulinkers are particularly useful for antibody-dendrimer conjugation. Inaddition, many tumor cells contain surface lectins that bind tooligosaccharides, with specific recognition arising chiefly from theterminal carbohydrate residues of the latter (See, e.g., Sharon and Lis,Science 246:227 (1989)). Attaching appropriate monosaccharides tononglycosylated proteins such as BSA provides a conjugate that binds totumor lectin much more tightly than the free monosaccharide (See, e.g.,Monsigny et al., Biochemie 70:1633 (1988)).

Mannosylated PAMAM dendrimers bind mannoside-binding lectin up to 400more avidly than monomeric mannosides (See, e.g., Page and Roy,Bioconjugate Chem., 8:714 (1997)). Sialylated dendrimers and otherdendritic polymers bind to and inhibit a variety of sialate-bindingviruses both in vitro and in vivo. By conjugating multiplemonosaccharide residues (e.g., α-galactoside, for galactose-bindingcells) to dendrimers, polyvalent conjugates are created with a highaffinity for the corresponding type of tumor cell. The attachmentreaction are easily carried out via reaction of the terminal amines withcommercially-available α-galactosidyl-phenylisothiocyanate. The smallsize of the carbohydrates allows a high concentration to be present onthe dendrimer surface.

Related to the targeting approaches described above is the“pretargeting” approach (See e.g., Goodwin and Meares, Cancer (suppl.)80:2675 (1997)). An example of this strategy involves initial treatmentof a subject with conjugates of tumor-specific monoclonal antibodies andstreptavidin. Remaining soluble conjugate is removed from thebloodstream with an appropriate biotinylated clearing agent. When thetumor-localized conjugate is all that remains, a radiolabeled,biotinylated agent is introduced, which in turn localizes at the tumorsites by the strong and specific biotin-streptavidin interaction. Thus,the radioactive dose is maximized in dose proximity to the cancer cellsand minimized in the rest of the body where it can harm healthy cells.

It has been shown that if streptavidin molecules bound to a polystyrenewell are first treated with a biotinylated dendrimer, and thenradiolabeled streptavidinis introduced, up to four of the labeledstreptavidin molecules are bound per polystyrene-bound streptavidin(See, e.g., Wilbur et al., Bioconjugate Chem., 9:813 (1998)). Thus,biotinylated dendrimers may be used in the methods of the presentinvention, acting as a polyvalent receptor for the radiolabel in vivo,with a resulting amplification of the radioactive dosage per boundantibody conjugate. In the preferred embodiments of the presentinvention, one or more multiply-biotinylated module(s) on the clustereddendrimer presents a polyvalent target for radiolabeled or boronated(See, e.g., Barth et al., Cancer Investigation 14:534 (1996)) avidin orstreptavidin, again resulting in an amplified dose of radiation for thetumor cells.

Dendrimers may also be used as clearing agents by, for example,partially biotinylating a dendrimer that has a polyvalent galactose ormannose surface. The conjugate-clearing agent complex would then have avery strong affinity for the corresponding hepatocyte receptors.

In other embodiments of the present invention, an enhanced permeabilityand retention (EPR) method is used in targeting. The enhancedpermeability and retention (EPR) effect is a more “passive” way oftargeting tumors (See, e.g., Duncan and Sat, Ann. Oncol., 9:39 (1998)).The EPR effect is the selective concentration of macromolecules andsmall particles in the tumor microenvironment, caused by thehyperpermeable vasculature and poor lymphatic drainage of tumors. Thedendrimer compositions of the present invention provide ideal polymersfor this application, in that they are relatively rigid, of narrowpolydispersity, of controlled size and surface chemistry, and haveinterior “cargo” space that can carry and then release antitumor drugs.In fact, PAMAM dendrimer-platinates have been shown to accumulate insolid tumors (Pt levels about 50 times higher than those obtained withcisplatin) and have in vivo activity in solid tumor models for whichcisplatin has no effect (See, e.g., Malik et al., Proc. Int'l. Symp.Control. Rel. Bioact. Mater., 24:107 (1997) and Duncan et al., PolymerPreprints 39:180 (1998)).

In some embodiments of the present invention, the preparation of PAMAMdendrimers is performed according to a typical divergent (building upthe macromolecule from an initiator core) synthesis. It involves atwo-step growth sequence that includes of a Michael addition of aminogroups to the double bond of methyl acrylate (MA) followed by theamidation of the resulting terminal carbomethoxy, —(CO₂ CH₃) group, withethylenediamine (EDA).

In the first step of this process, ammonia is allowed to react under aninert nitrogen atmosphere with MA (molar ratio: 1:4.25) at 47° C. for 48hours. The resulting compound is referred to as generation=0, thestar-branched PAMAM tri-ester. The next step involves reacting thetri-ester with an excess of EDA to produce the star-branched PAMAMtri-amine (G=O). This reaction is performed under an inert atmosphere(nitrogen) in methanol and requires 48 hours at 0° C. for completion.Reiteration of this Michael addition and amidation sequence producesgeneration=1.

Preparation of this tri-amine completes the first full cycle of thedivergent synthesis of PAMAM dendrimers. Repetition of this reactionsequence results in the synthesis of larger generation (G=1-5)dendrimers (i.e., ester- and amine-terminated molecules, respectively).For example, the second iteration of this sequence produces generation1, with an hexa-ester and hexa-amine surface, respectively. The samereactions are performed in the same way as for all subsequentgenerations from 1 to 9, building up layers of branch cells giving acore-shell architecture with precise molecular weights and numbers ofterminal groups as shown above. Carboxylate-surfaced dendrimers can beproduced by hydrolysis of ester-terminated PAMAM dendrimers, or reactionof succinic anhydride with amine-surfaced dendrimers (e.g., fullgeneration PAMAM, POPAM or POPAM-PAMAM hybrid dendrimers).

Various dendrimers can be synthesized based on the core structure thatinitiates the polymerization process. These core structures dictateseveral important characteristics of the dendrimer molecule such as theoverall shape, density, and surface functionality (See, e.g., Tomalia etal., Angew. Chem. Int. Ed. Engl., 29:5305 (1990)). Spherical dendrimersderived from ammonia possess trivalent initiator cores, whereas EDA is atetra-valent initiator core. Recently, rod-shaped dendrimers have beenreported which are based upon linear poly(ethyleneimine) cores ofvarying lengths the longer the core, the longer the rod (See, e.g., Yinet al., J. Am. Chem. Soc., 120:2678 (1998)).

In some embodiments, dendrimers of the present invention comprise aprotected core diamine. In some embodiments, the protected initiatorcore diamine is NH2-(CH2)_(n)-NHPG, (n=1-10). In other embodiments, theintitor core is selected from the group comprising, but not limited to,NH2-(CH2)_(n)-NH2 (n=1-10), NH2-((CH2)_(n)NH2)₃ (n=1-10), orunsubstituted or substituted 1,2-; 1,3-; or1,4-phenylenedi-n-alkylamine, with a monoprotected diamine (e.g.,NH2-(CH2)_(n)-NHPG) used during the amide formation of each generation.In these approaches, the protected diamine allows for the large scaleproduction of dendrimers without the production of non-uniformnanostructures that can make characterization and analysis difficult. Bylimiting the reactivity of the diamine to only one terminus, theopportunities of dimmer/polymer formation and intramolecular reactionsare obviated without the need of employing large excesses of diamine.The terminus monoprotected intermediates can be readily purified sincethe protecting groups provide suitable handle for productivepurifications by classical techniques like crystallization and orchromatography.

The protected intermediates can be deprotected in a deprotection step,and the resulting generation of the dendrimer subjected to the nextiterative chemical reaction without the need for purification. Theinvention is not limited to a particular protecting group. Indeed avariety of protecting groups are contemplated including, but not limitedto, t-butoxycarbamate (N-t-Boc), allyloxycarbamate (N-Alloc),benzylcarbamate (N-Cbz), 9-fluorenylmethylcarbamate (FMOC), orphthalimide (Phth). In preferred embodiments of the present invention,the protecting group is benzylcarbamate (N-Cbz). N-Cbz is ideal for thethe present invention since it alone can be easily cleaved under“neutral” conditions by catalytic hydrogenation (Pd/C) without resortingto strongly acidic or basic conditions needed to remove an F-MOC group.The use of protected monomers finds particular use in high through-putproduction runs because a lower amount of monomer can be used, reducingproduction costs.

The dendrimers may be characterized for size and uniformity by anysuitable analytical techniques. These include, but are not limited to,atomic force microscopy (AFM), electrospray-ionization massspectroscopy, MALDI-TOF mass spectroscopy, ¹³C nuclear magneticresonance spectroscopy, high performance liquid chromatography (HPLC)size exclusion chromatography (SEC) (equipped with multi-angle laserlight scattering, dual UV and refractive index detectors), capillaryelectrophoresis and get electrophoresis. These analytical methods assurethe uniformity of the dendrimer population and are important in thequality control of dendrimer production for eventual use in in vivoapplications. Most importantly, extensive work has been performed withdendrimers showing no evidence of toxicity when administeredintravenously (Roberts et al., J. Biomed. Mater. Res., 30:53 (1996) andBoume et al., J. Magnetic Resonance Imaging, 6:305 (1996)).

In some embodiments of the present invention, the dendrimer conjugatescomprise transgenes for delivery and expression to a target cell ortissue, in vitro, ex vivo, or in vivo. In such embodiments, rather thancontaining the actual protein, the dendrimer complex comprises anexpression vector construct containing, for example, a heterologous DNAencoding a gene of interest and the various regulatory elements thatfacilitate the production of the particular protein of interest in thetarget cells.

In some embodiments, the gene is a therapeutic gene that is used, forexample, to treat cancer, to replace a defective gene, or a marker orreporter gene that is used for selection or monitoring purposes. In thecontext of a gene therapy vector, the gene may be a heterologous pieceof DNA. The heterologous DNA may be derived from more than one source(i.e., a multigene construct or a fusion protein). Further, theheterologous DNA may include a regulatory sequence derived from onesource and the gene derived from a different source.

Tissue-specific promoters may be used to effect transcription inspecific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate. Similarly, promoters may be used to target gene expression inother tissues (e.g., insulin, elastin amylase, pdr-1, pdx-1 andglucokinase promoters target to the pancreas; albumin PEPCK, HBVenhancer, alpha fetoproteinapolipoprotein C, alpha-1 antitrypsin,vitellogenin, NF-AB and transthyretin promoters target to the liver;myosin H chain, muscle creatine kinase, dystrophin, calpain p94,skeletal alpha-actin, fast troponin 1 promoters target to skeletalmuscle; keratin promoters target the skin; sm22 alpha; SM-.alpha.-actinpromoters target smooth muscle; CFTR; human cytokeratin 18 (K18);pulmonary surfactant proteins A, B and Q CC-10; P1 promoters target lungtissue; endothelin-1; E-selectin; von Willebrand factor; KDR/flk-1target the endothelium; tyrosinase targets melanocytes).

The nucleic acid may be either cDNA or genomic DNA. The nucleic acid canencode any suitable therapeutic protein. Preferably, the nucleic acidencodes a tumor suppressor, cytokine, receptor, inducer of apoptosis, ordifferentiating agent. The nucleic acid may be an antisense nucleicacid. In such embodiments, the antisense nucleic acid may beincorporated into the nanodevice of the present invention outside of thecontext of an expression vector.

In preferred embodiments, the nucleic acid encodes a tumor suppressor,cytokines, receptors, or inducers of apoptosis. Suitable tumorsuppressors include BRCA1, BRCA2, C-CAM, p16, p211 p53, p73, or Rb.Suitable cytokines include GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,β-interferon, γ-interferon, or TNF. Suitable receptors include CFTR,EGFR, estrogen receptor, IL-2 receptor, or VEGFR. Suitable inducers ofapoptosis include AdE1B, Bad, Bak, Bax, Bid, Bik, Bim, Harakiri, orICE-CED3 protease.

In some embodiments, more than one administration of the dendrimerconjugates of the present invention or the other agent are utilized.Various combinations may be employed, where the dendrimer is “A” (e.g.,comprising a pain relief agent) and the other agent is “B” (e.g.,comprising a pain relief agent antagonist), as exemplified below:

A/B/A, B/A/B, B/B/A, A/A/B, B/A/A, A/B/B, B/B/B/A, B/B/A/B, A/A/B/B,A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, B/B/B/A, A/A/A/B, B/A/A/A,A/B/A/A, A/A/B/A, A/B/B/B, B/A/B/B, B/B/A/B.

Other combinations are contemplated.

Other factors that may be used in combination therapy with the dendrimerconjugates of the present invention include, but are not limited to,factors that cause DNA damage such as gamma-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-irradiation. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 weeks), to singledoses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes varywidely, and depend on the half-life of the isotope, the strength andtype of radiation emitted, and the uptake by the neoplastic cells. Theskilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

In preferred embodiments of the present invention, the regional deliveryof the dendrimer conjugates to patients with cancers is utilized tomaximize the therapeutic effectiveness of the delivered agent.Similarly, the chemo- or radiotherapy may be directed to particular,affected region of the subjects body. Alternatively, systemic deliveryof the immunotherapeutic composition and/or the agent may be appropriatein certain circumstances, for example, where extensive metastasis hasoccurred.

In addition to combining the dendrimer conjugates with chemo- andradiotherapies, it also is contemplated that traditional gene therapiesare used. For example, targeting of p53 or p16 mutations along withtreatment of the dendrimer conjugates provides an improved anti-cancertreatment. The present invention contemplates the co-treatment withother tumor-related genes including, but not limited to, p21, Rb, APC,DCC, NF-I, NF-2, BCRA2, p16, FHIT, WT-I, MEN-I, MEN-II, BRCA1, VHL, FCC,MCC, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl, andabl.

In vivo and ex vivo treatments are applied using the appropriate methodsworked out for the gene delivery of a particular construct for aparticular subject. For example, for viral vectors, one typicallydelivers 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹ or1×10¹² infectious particles to the patient. Similar figures may beextrapolated for liposomal or other non-viral formulations by comparingrelative uptake efficiencies.

An attractive feature of the present invention is that the therapeuticcompositions may be delivered to local sites in a patient by a medicaldevice. Medical devices that are suitable for use in the presentinvention include known devices for the localized delivery oftherapeutic agents. Such devices include, but are not limited to,catheters such as injection catheters, balloon catheters, double ballooncatheters, microporous balloon catheters, channel balloon catheters,infusion catheters, perfusion catheters, etc., which are, for example,coated with the therapeutic agents or through which the agents areadministered; needle injection devices such as hypodermic needles andneedle injection catheters; needleless injection devices such as jetinjectors; coated stents, bifurcated stents, vascular grafts, stentgrafts, etc.; and coated vaso-occlusive devices such as wire coils.

Exemplary devices are described in U.S. Pat. Nos. 5,935,114; 5,908,413;5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719;5,851,228; 5,843,089; 5,800,519; 5,800,508; 5,800,391; 5,354,308;5,755,722; 5,733,303; 5,866,561; 5,857,998; 5,843,003; and 5,933,145;the entire contents of which are incorporated herein by reference.Exemplary stents that are commercially available and may be used in thepresent application include the RADIUS (SCIMED LIFE SYSTEMS, Inc.), theSYMPHONY (Boston Scientific Corporation), the Wallstent (SchneiderInc.), the PRECEDENT II (Boston Scientific Corporation) and the NIR(Medinol Inc.). Such devices are delivered to and/or implanted at targetlocations within the body by known techniques.

In some embodiments, the therapeutic complexes of the present inventioncomprise a photodynamic compound and a targeting agent that isadministred to a patient. In some embodiments, the targeting agent isthen allowed a period of time to bind the “target” cell (e.g. about 1minute to 24 hours) resulting in the formation of a target cell-targetagent complex. In some embodiments, the therapeutic complexes comprisingthe targeting agent and photodynamic compound are then illuminated(e.g., with a red laser, incandescent lamp, X-rays, or filteredsunlight). In some embodiments, the light is aimed at the jugular veinor some other superficial blood or lymphatic vessel. In someembodiments, the singlet oxygen and free radicals diffuse from thephotodynamic compound to the target cell (e.g. cancer cell or pathogen)causing its destruction.

Where clinical applications are contemplated, in some embodiments of thepresent invention, the dendrimer conjugates are prepared as part of apharmaceutical composition in a form appropriate for the intendedapplication. Generally, this entails preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals. However, in some embodiments of thepresent invention, a straight dendrimer formulation may be administeredusing one or more of the routes described herein.

In preferred embodiments, the dendrimer conjugates are used inconjunction with appropriate salts and buffers to render delivery of thecompositions in a stable manner to allow for uptake by target cells.Buffers also are employed when the dendrimer conjugates are introducedinto a patient. Aqueous compositions comprise an effective amount of thedendrimer conjugates to cells dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. Except insofar as any conventional media or agent is incompatiblewith the vectors or cells of the present invention, its use intherapeutic compositions is contemplated. Supplementary activeingredients may also be incorporated into the compositions.

In some embodiments of the present invention, the active compositionsinclude classic pharmaceutical preparations. Administration of thesecompositions according to the present invention is via any common routeso long as the target tissue is available via that route. This includesoral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection.

The active dendrimer conjugates may also be administered parenterally orintraperitoneally or intratumorally. Solutions of the active compoundsas free base or pharmacologically acceptable salts are prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

In some embodiments, a therapeutic agent is released from dendrimerconjugates within a target cell (e.g., within an endosome). This type ofintracellular release (e.g., endosomal disruption of alinker-therapeutic conjugate) is contemplated to provide additionalspecificity for the compositions and methods of the present invention.In some embodiments, the dendrimer conjugates of the present inventioncontain between 100-150 primary amines on the surface. Thus, the presentinvention provides dendrimers with multiple (e.g., 100-150) reactivesites for the conjugation of linkers and/or functional groupscomprising, but not limited to, therapeutic agents, targeting agents,imaging agents and biological monitoring agents.

The compositions and methods of the present invention are contemplatedto be equally effective whether or not the dendrimer conjugates of thepresent invention comprise a fluorescein (e.g. FITC) imaging agent.Thus, each functional group present in a dendrimer composition is ableto work independently of the other functional groups. Thus, the presentinvention provides dendrimer conjugates that can comprise multiplecombinations of targeting, therapeutic, imaging, and biologicalmonitoring functional groups. Additionally, in some embodiments, eachfunctional group (e.g., therapeutic agents, targeting agents, imagingagents and biological monitoring agents) present in a dendrimercomposition can function together with one or more of the functionalgroups (e.g., cooperative binding of multiple targeting ligands).

The present invention also provides a very effective and specific methodof delivering molecules (e.g., therapeutic and imaging functionalgroups) to the interior of target cells (e.g., cancer cells). Thus, insome embodiments, the present invention provides methods of therapy thatcomprise or require delivery of molecules into a cell in order tofunction (e.g., delivery of genetic material such as siRNAs).

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The carrier may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial anantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it may be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, dendrimer conjugates are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution is suitably buffered, if necessary,and the liquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). In some embodiments of the present invention, the activeparticles or agents are formulated within a therapeutic mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses may be administered.

Additional formulations that are suitable for other modes ofadministration include vaginal suppositories and pessaries. A rectalpessary or suppository may also be used. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or the urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries areusually globular or oviform and weighing about 5 g each. Vaginalmedications are available in a variety of physical forms, e.g., creams,gels or liquids, which depart from the classical concept ofsuppositories. In addition, suppositories may be used in connection withcolon cancer. The dendrimer conjugates also may be formulated asinhalants for the treatment of lung cancer and such like.

In some embodiments of the present invention methods and compositionsare provided for the treatment of tumors in cancer therapy. It iscontemplated that the present therapy can be employed in the treatmentof any cancer for which a specific signature has been identified orwhich can be targeted. Cell proliferative disorders, or cancers,contemplated to be treatable with the methods of the present inventioninclude human sarcomas and carcinomas, including, but not limited to,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,Ewing's tumor, lymphangioendotheliosarcoma, synovioma, mesothelioma,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilns' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrbm's macroglobulinemia, and heavychain disease.

In some embodiments of the present invention, methods and compositionsare provided for the treatment of inflammatory diseases (e.g.,dendrimers conjugated with therapeutic agents configured for treatinginflammatory diseases). Inflammatory diseases include but are notlimited to arthritis, rheumatoid arthritis, psoriatic arthritis,osteoarthritis, degenerative arthritis, polymyalgia rheumatic,ankylosing spondylitis, reactive arthritis, gout, pseudogout,inflammatory joint disease, systemic lupus erythematosus, polymyositis,and fibromyalgia. Additional types of arthritis include achillestendinitis, achondroplasia, acromegalic arthropathy, adhesivecapsulitis, adult onset Still's disease, anserine bursitis, avascularnecrosis, Behcet's syndrome, bicipital tendinitis, Blount's disease,brucellar spondylitis, bursitis, calcaneal bursitis, calciumpyrophosphate 73mperfect (CPPD), crystal deposition disease, Caplan'ssyndrome, carpal tunnel syndrome, chondrocalcinosis, chondromalaciapatellae, chronic synovitis, chronic recurrent multifocal osteomyelitis,Churg-Strauss syndrome, Cogan's syndrome, corticosteroid-inducedosteoporosis, costosternal syndrome, CREST syndrome, cryoglobulinemia,degenerative joint disease, dermatomyositis, diabetic finger sclerosis,diffuse idiopathic skeletal hyperostosis (DISH), discitis, discoid lupuserythematosus, drug-induced lupus, Duchenne's muscular dystrophy,Dupuytren's contracture, Ehlers-Danlos syndrome, enteropathic arthritis,epicondylitis, erosive inflammatory osteoarthritis, exercise-inducedcompartment syndrome, Fabry's disease, familial Mediterranean fever,Farber's lipogranulomatosis, Felty's syndrome, Fifth's disease, flatfeet, foreign body synovitis, Freiberg's disease, fungal arthritis,Gaucher's disease, giant cell arteritis, gonococcal arthritis,Goodpasture's syndrome, granulomatous arteritis, hemarthrosis,hemochromatosis, Henoch-Schonlein purpura, Hepatitis B surface antigendisease, hip dysplasia, Hurler syndrome, hypermobility syndrome,hypersensitivity vasculitis, hypertrophic osteoarthropathy, immunecomplex disease, impingement syndrome, Jaccoud's arthropathy, juvenileankylosing spondylitis, juvenile dermatomyositis, juvenile rheumatoidarthritis, Kawasaki disease, Kienbock's disease, Legg-Calve-Perthesdisease, Lesch-Nyhan syndrome, linear scleroderma, lipoiddermatoarthritis, Lofgren's syndrome, Lyme disease, malignant synovioma,Marfan's syndrome, medial plica syndrome, metastatic carcinomatousarthritis, mixed connective tissue disease (MCTD), mixedcryoglobulinemia, mucopolysaccharidosis, multicentricreticulohistiocytosis, multiple epiphyseal dysplasia, mycoplasmalarthritis, myofascial pain syndrome, neonatal lupus, neuropathicarthropathy, nodular panniculitis, ochronosis, olecranon bursitis,Osgood-Schlatter's disease, osteoarthritis, osteochondromatosis,osteogenesis imperfecta, osteomalacia, osteomyelitis, osteonecrosis,osteoporosis, overlap syndrome, pachydermoperiostosis Paget's disease ofbone, palindromic rheumatism, patellofemoral pain syndrome,Pellegrini-Stieda syndrome, pigmented villonodular synovitis, piriformissyndrome, plantar fasciitis, polyarteritis nodos, Polymyalgia rheumatic,polymyositis, popliteal cysts, posterior tibial tendinitis, Pott'sdisease, prepatellar bursitis, prosthetic joint infection,pseudoxanthoma elasticum, psoriatic arthritis, Raynaud's phenomenon,reactive arthritis/Reiter's syndrome, reflex sympathetic dystrophysyndrome, relapsing polychondritis, retrocalcaneal bursitis, rheumaticfever, rheumatoid vasculitis, rotator cuff tendinitis, sacroiliitis,salmonella osteomyelitis, sarcoidosis, saturnine gout, Scheuermann'sosteochondritis, scleroderma, septic arthritis, seronegative arthritis,shigella arthritis, shoulder-hand syndrome, sickle cell arthropathy,Sjogren's syndrome, slipped capital femoral epiphysis, spinal stenosis,spondylolysis, staphylococcus arthritis, Stickler syndrome, subacutecutaneous lupus, Sweet's syndrome, Sydenham's chorea, syphiliticarthritis, systemic lupus erythematosus (SLE), Takayasu's arteritis,tarsal tunnel syndrome, tennis elbow, Tietse's syndrome, transientosteoporosis, traumatic arthritis, trochanteric bursitis, tuberculosisarthritis, arthritis of Ulcerative colitis, undifferentiated connectivetissue syndrome (UCTS), urticarial vasculitis, viral arthritis,Wegener's granulomatosis, Whipple's disease, Wilson's disease, andyersinial arthritis.

In some embodiments, the dendrimer conjugates configured for treatinginflammatory disorders (e.g., rheumatoid arthritis) are co-administeredto a subject (e.g., a human suffering from an inflammatory disorder) atherapeutic agent configured for treating inflammatory disorders (e.g.,rheumatoid arthritis). Examples of such agents include, but are notlimited to, disease-modifying antirheumatic drugs (e.g., leflunomide,methotrexate, sulfasalazine, hydroxychloroquine), biologic agents (e.g.,rituximab, infliximab, etanercept, adalimumab, golimumab), nonsteroidalanti-inflammatory drugs (e.g., ibuprofen, celecoxib, ketoprofen,naproxen, piroxicam, diclofenac), analgesics (e.g., acetaminophen,tramadol), immunomodulators (e.g., anakinra, abatacept), andglucocorticoids (e.g., prednisone, methylprednisone).

The present invention also includes methods involving co-administrationof the multifunctional dendrimers and components thereof describedherein with one or more additional active agents. Indeed, it is afurther aspect of this invention to provide methods for enhancing priorart therapies and/or pharmaceutical compositions by co-administeringmultifunctional dendrimers of this invention. In co-administrationprocedures, the agents may be administered concurrently or sequentially.In some embodiments, the multifunctional dendrimers described herein areadministered prior to the other active agent(s). The agent or agents tobe co-administered depends on the type of condition being treated. Forexample, when the condition being treated is cancer, the additionalagent can be a chemotherapeutic agent or radiation. The additionalagents to be co-administered, such as anticancer agents, can be any ofthe well-known agents in the art, including, but not limited to, thosethat are currently in clinical use. The determination of appropriatetype and dosage of radiation treatment is also within the skill in theart or can be determined with relative ease.

Where clinical applications are contemplated, in some embodiments of thepresent invention, the dendrimer conjugates are prepared as part of apharmaceutical composition in a form appropriate for the intendedapplication. Generally, this entails preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals. However, in some embodiments of thepresent invention, a straight dendrimer formulation may be administeredusing one or more of the routes described herein. It is contemplatedthat the present therapy can be employed in the treatment of anypathogenic disease for which a specific signature has been identified orwhich can be targeted for a given pathogen. Examples of pathogenscontemplated to be treatable with the methods of the present inventioninclude, but are not limited to, Legionella peomophilia, Mycobacteriumtuberculosis, Clostridium tetani, Hemophilus influenzae, Neisseriagonorrhoeae, Treponema pallidum, Bacillus anthracis, Vibrio cholerae,Borrelia burgdorferi, Cornebacterium diphtheria, Staphylococcus aureus,human papilloma virus, human immunodeficiency virus, rubella virus,polio virus, and the like.

Experimental

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1

Experiments were conducted during development of embodiments of theinvention in order to analyze and characterize various schemes forgenerating dendrimer conjugates wherein a dendrimer is conjugated to oneor more linkers that comprise multiple sites for binding (e.g., covalentbinding) moieties. A drug releasing mechanism for esterase sensitivelinker-dendrimer conjugates was analyzed (See e.g., FIG. 10). In someembodiments, once the ester bond is cleaved (e.g., by esterases (e.g.,present at a target site (e.g., intrinsic to the target))), irreversibledecomposition of the linkers leads to release of drug and/or therapeuticagent (e.g., at the target site).

Three elimination linkers (See FIG. 11, A-C) designed for esterasetriggered cleavage were synthesized. In some embodiments, the linkersare conjugated to a therapeutic agent and/or to a dendrimer (e.g., G5dendrimer).

Example 2 Synthesis of Esterase Sensitive Linker 11A

A synthesis scheme of a dendrimer (e.g., G5 PAMAM dendrimer) conjugatedto a therapeutic agent (e.g., TAXOL) with an esterase sensitive linker(esterase sensitive elimination linker 11A) is shown below.

Step 1:

A 50 mL solution of Boc-β-alanine (500 mg, 2.64 mmol), EDC (506 mg, 2.64mmol), and DMAP (322 mg, 2.64 mmol) in methylene chloride was stirred at0° C. for 20 min. 4-Hydroxybenzaldehyde (323 mg, 2.64 mmol) was thenadded slowly. The reaction mixture was stirred at 0° C. for 2 hourbefore it was warmed to RT and continued for over night. The reactionmixture was then diluted with EtOAc and H₂O and extractive work up togive a crude product which was purified by silica gel chromatography toafford a clear oil (712 mg, 92%).

MS (EI) m/e=294 (M+1)

Step 2:

The aldehyde 1 (775 mg, 2.64 mmol) was dissolved in 40 mL of dry THF andwas cooled to 0° C. Boran in THF (1N solution, 2.64 mL) was addeddropwise. The reaction mixture was for 2 h. MeOH (5 mL) was added slowlyand the reaction mixture was warmed to RT in 1 h. Solvent was evaporatedand the product was purified by chromatograph to afford the product as awhite solid (625 mg, 80%).

MS (EI) m/e=296 (M+1)

Step 3:

The benzyl alcohol 2 (456 mg, 1.54 mmol) and p-nitrobenzyl chloroformate(934 mg, 4.63 mmol) were dissolved in 20 mL of methylene chloride.Pyridine (0.42 mL, 5.19 mmol) was added. White precipitate was formedduring the addition process. The reaction mixture was stirred at RT overnight. The reaction mixture was then diluted with EtOAc and water.Layers were separated and the aqueous layer was extracted with EtOAc×3.Combined organic solution was washed with 1N HCl, sat'd NaHCO₃ andbrine. The crude mixture was purified by silica gel chromatographyeluting with 15-25% EtOAc in Hexanes to afford the product as clear oil(520 mg, 73%).

MS (EI) m/e=461 (M+1)

Step 4:

Taxol (9.3 mg, 0.0103575 mmol) and 3 (4.75 mg, 0.0103575 mmol) weredissolved in dry methylene chloride (1 mL). A solution of DMAP (2.5 mg,0.02715 mmol) in methylene chloride (1 mL) was added dropwise at roomtemperature. After addition, a light yellow color appeared. The reactionmixture was allowed to stir at room temperature for 3 hours when TLCindicated the reaction was complete. The reaction mixture was extractedwith methylene chloride and water. The organic layers were combined anddried over MgSO₄ Solvent was evaporated after filtration. The residuewas purified by column chromatography (silica gel, EtOAc:Hexanes 1:1)and pure product (10 mg, yield 82%) was obtained.

MS (EI) m/e=1197.5 (M+Na).

Step 5:

Taxol-linker conjugate 4 (10.0 mg, 0.008514 mmol) was dissolved inmethylene chloride (1 mL). To above solution was added TFA (120 μL). Thereaction mixture was stirred at room temperature and was checked withTLC until the reaction was complete in 20 minutes. The solvent wasevaporated and the residue was purified by column chromatography (silicagel, CH₂Cl₂:MeOH 10:1). Product 5 was isolated as a white solid (8.0 mg,yield 87.4%).

MS (EI) m/e=1075.4 (M+H).

Step 6:

G5-Ac-FI-FA-COOH (6), prepared as reported previously, (15.3 mg,0.0004636 mmol) was dissolved in H₂O (5.2 mL), EDM (10.6 mg, 0.03488mmol) was added. The reaction mixture was stirred for 2 hour at roomtemperature. A solution of 5 (10 mg, 0.0093 mmol) in DMF (4.3 mL) andDMSO (3.4 mL) was added dropwise. The reaction was allowed to stir atroom temperature for three days. The solvent was removed by membranefiltration through a 10,000 MWCO membrane. The residue was furtherpurified by passing through a Sephdex G-25 column and extensively washedwith PBS buffer and water. Lyophilization gave final product 7 as orangecolored solid (15.1 mg, yield 90%).

Example 3 Synthesis of Esterase Sensitive Linker 11B

A synthesis scheme of a dendrimer (e.g., G5 PAMAM dendrimer) conjugatedto a therapeutic agent (e.g., Taxol) with an esterase sensitive linker(esterase sensitive elimination linker 11B) is shown below.

Step 1:

A mixture of 5-formyl-2-hydroxybenzoic acid 8 (1.66 g, 10 mmol),mono-Boc-ethylene diamine (1.60 g, 10 mmol), EDC methiodide (2.97 g, 10mmol), and HOBT (1.35 g, 10 mmol) was dissolved in 40 mL of DMF at 0 °C. The solution was stirred at this temperature for 1 h before it waswarmed to RT. Stirring was continued for over night. The orange-yellowcolored reaction mixture was cooled to 0° C. Triethylamine (2.8 mL, 20mmol) was added followed by pivaloyl chloride (2.5 mL, 20 mmol). Thereaction mixture was stirred for 2 hours and it was quenched by additionof 50 ml of water. EtOAc (200 mL) was added and the layers wereseparated. The aqueous layer was extracted with EtOAc×3. The combinedorganics was washed with 1N HCl, saturated NaHCO₃ solution, and brinesequentially and was dried with MgSO₄. After solvent was evaporated, theresidue was purified by silica gel chromatography to afford the product9 as a pale yellow solid (3.33 g, 85% 2 steps).

MS (EI) m/e=xxx (M+1)

Step 2:

The aldehyde 9 (2.04 g, 5.20 mmol) was dissolved in 60 mL of dry THF andwas cooled to 0° C. Boran in THF (1N solution, 5.46 mL) was addeddropwise. The reaction mixture was for 2 h. MeOH (10 mL) was addedslowly and the reaction mixture was warmed to RT in 2 h. Solvent wasevaporated and the product was purified by chromatograph to afford theproduct as a white solid (2.02 g, 98%).

MS (EI) m/e=xxx (M+1)

Step 3:

The benzyl alcohol 10 (1.185 g, 3.0 mmol) and p-nitrobenzylchloroformate (908 mg, 4.50 mmol) were dissolved in 30 mL of methylenechloride. Pyridine (0.49 mL, 6.0 mmol) was added. White precipitate wasformed during the addition process. The reaction mixture was stirred atRT over night. The reaction mixture was then diluted with EtOAc andwater. Layers were separated and the aqueous layer was extracted withEtOAc×3. Combined organic solution was washed with 1N HCl, sat'd NaHCO₃and brine. The crude mixture was purified by silica gel chromatographyeluting with 15-25% EtOAc in Hexanes to afford the product 11 as whitesolid (1.38 g, 82%).

MS (EI) m/e=560 (M+1)

Step 4:

In a 5 mL round bottle flask, taxol (20 mg, 0.02227 mmol) and linker 11(35.8 mg, 0.02810 mmol) were dissolved in dry methylene chloride (2 mL).A solution of DMAP (5.7 mg, 0.04666 mmol) in methylene chloride (1 mL)was added dropwise at room temperature. After addition, a light yellowappeared. The reaction mixture was allowed to stir at room temperaturefor 3 hours. The reaction was monitored with TLC until the reaction wascomplete. The reaction mixture was extracted with methylene chloride andwater. The organic layer was collected and dried over MgSO₄ andevaporated. The residue was purified by column chromatography (silicagel, AcOEt:Hexanes 1:1) and pure product (25.3 mg, yield 89%) wasobtained.

MS (EI) m/e=1296.5(M+Na).

Step 5:

To the taxol-linker conjugate 12 (12 mg, 0.009426 mmol) in methylenechloride (1 mL) was added TFA (120 μL). The reaction mixture was stirredat room temperature for 20 minutes and was checked with TLC until thereaction was complete. The solvent was evaporated and the residue waspurified by column chromatography (silica gel, CH₂Cl₂:MeOH 10:1).Product 13 was isolated as a white solid (9.8 mg, yield 88%).

MS (EI) m/e=1174.5 (M+H).

Step 6:

G5-Ac-FI-FA-COOH (6), prepared as reported previously, (13.8 mg,0.00041818 mmol) was dissolved in H₂O (5.2 mL), EDM (9.32 mg, 0.03136mmol) was added. The reaction mixture was stirred for 2 hour at roomtemperature. A solution of 13 (9.8 mg, 0.0093 mmol) in DMF (4.3 mL) andDMSO (3.4 mL) was added dropwise. The reaction was allowed to stir atroom temperature for three days. The solvent was removed by membranefiltration through a 10,000 MWCO membrane. The residue was furtherpurified by passing through a Sephdex G-25 column and extensively washedwith PBS buffer and water. Lyophilization gave final product 14 asorange colored solid (15.1 mg, yield 87%).

Example 4 Synthesis of Esterase Sensitive Linker 11C

A synthesis scheme of a dendrimer (e.g., G5 PAMAM dendrimer) conjugatedto a therapeutic agent (e.g., Taxol) with an esterase sensitive linker(esterase sensitive elimination linker 11C) is shown below.

Example 5 Additional Self-Immorlative Linkers

The present invention is not limited by the type of self-immorlativelinkers utilized. For example, cyclization based linkers can be used.Although a mechanism is not necessary to practice the present inventionand the present invention is not limited to any particular mechanism, insome embodiments, a mechanism as shown below is utilized in a conjugateof the present invention:

Thus, in some embodiments, the present invention provides synthesis ofdendrimer conjugates utilizing cyclization linkers (e.g., designed asesterase cleavage substrates) as shown below:

In some embodiments, the present invention provides syntheses of linkersC and D as shown below.

Example 6 Characterization of Dendrimer Conjugates

Experiments were conducted during development of embodiments of theinvention in order to characterize release of drug from a dendrimerconjugate comprising a linker-drug component. The linker-drug componentswere characterized under esterase incubation conditions, utilizing HPLCas an analytical tool to monitor drug release. This approach provides anassessment regarding structural influences of the linkers. For example,characteristics of drug release from a linker (e.g., in the absence of adendrimer) provides information regarding drug release from a linkerconjugated to a dendrimer.

For example, the experiments were conducted to characterize thefollowing two conjugates:

First Generation Linker-Drug(-Dendrimer) Conjugates

When incubated with pig liver esterase for 2 hours, conjugate B showedminimal release and conjugate A showed ˜11% release. Furthermore,conjugate A showed around 40% release at 24 h (See, e.g., FIG. 12).

Example 7 Second Generation Linkers

Characterization of linkers as described in Example 6 indicated thatsteric hindrance issues were inhibiting release of a therapeutic fromthe conjugates (e.g., due, in some embodiments, to inaccessibility ofesterase to the linker). Based on this data, alternative approaches weregenerated and characterized. For example, in some embodiments, in orderto relieve steric hindrance, lengths of the linkers were extended. Thus,the present invention provides additional, “second generation” linkersas described below.

Second Generation Linker-Drug(-Dendrimer) Conjugates Rational

Thus, in some embodiments, the present invention provides conjugates andmethods of synthesizing and utilizing (e.g., therapeutically) the samewith extended linkages as shown in FIG. 13. In some embodiments thepresent invention provides conjugates as shown in FIGS. 14-16.

Example 8 Hypoxia Induced Linkers

The present invention also provides dendrimers comprising small moleculelinkers triggered by hypoxic environments (e.g., in and/or around cancercells). In some embodiments, a dendrimer of the present inventioncomprises a indolequinone linker. In some embodiments, a dendrimercomprising a hypoxia cleavable linker is generated according to thesynthesis scheme shown in FIG. 29. In some embodiments, a dendrimercomprising a hypoxia cleavable linker is generated according to thesynthesis scheme shown in FIG. 30. The present invention is not limitedto any particular mechanism of release of a therapeutic agent from adendrimer comprising a linker triggered by a hypoxic environment.Indeed, a variety of mechanisms are contemplated including, but notlimited to, a mechanism shown in FIG. 31.

Example 9 Synthesis and Conjugation of Locking Agent

For the synthesis of the locking module (tetrahydropyridinium,3-[[(3-carboxypropyl)amino]carbonyl]-1-methyl), γ-aminobutyric acid(GABA), benzyl ester is linked to an interconvertibletetrahydronicotinamide/quaternary nicotinamide salt structure that isable to participate in a re-dox type reaction. Thetetrahydronicotinamides are lipophilic stable compounds that readilyoxidize back to precursor quaternary salts by oxidase and peroxidaseenzymes in vivo. The synthesis of this compound is performed accordingto a modified literature procedure (see, e.g., Carelli, V., et al.,Bioorganic & Medicinal Chemistry Letters, 2003. 13(21): p. 3765-3769;herein incorporated by reference in its entirety) (Scheme 4). Reactionof the GABA ester with nicotinoyl chloride gives the correspondingnicotinamide derivative. The deprotection of the benzyl ester byhydrogenation using Pd/C catalyst yields the GABA derivative that istransformed to a quaternary salt by treatment with dimethyl-sulfate. Thepyridinium salt is reduced with Na₂S₂O₄ or electrochemically to give atetrahydronicotinamide derivative. This compound is conjugated invarying ratios to partially acetylated dendrimer using carbodimidecoupling to give the desired locking function to the dendrimerconjugate.

Example 10 Synthesis and Conjugation of Drug-Linkers Syntheses ofEsterase Sensitive Morphine-Linker for Conjugated to the CNS TargetingDendrimer

Prodrug approaches have been applied to Morphine in many studies inorder to improve solubility, absorption, tissue selectivity, and otherdrug delivery properties. Generally, either or both 3- and 6-hydroxylgroups are converted to an ester (see, e.g., Christrup, L. L., et al.,International Journal of Pharmaceutics, 1997. 154(2): p. 157-165;Drustrup, J., et al., International Journal of Pharmaceutics, 1991.71(1-2): p. 105-116; Groth, L., et al., International Journal ofPharmaceutics, 1997. 154(2): p. 149-155; Mignat, C., et al., Journal ofPharmaceutical Sciences, 1996. 85(7): p. 690-694; each hereinincorporated by reference in their entireties). The kinetics of esterenzymatic hydrolysis have been studied well. The present inventionprovides dendrimer conjugates wherein ester prodrugs are attached to adendrimer platform that possesses CNS targeting characteristics. Sincethe structural features of the ester significantly affect the rate ofhydrolysis, three different ester linkages between Morphine and thedendrimer are provdided. Serum esterase catalyzed drug release reactionsare carried out to find the desired hydrolysis profile.

Morphine-linker A, a 3-Aliphatic Acid Ester (see, e.g., Daniels, T. R.,et al., Clinical Immunology, 2006. 121(2): p. 159-176; Daniels, T. R.,et al., Clinical Immunology, 2006. 121(2): p. 144-158; Carelli, V., etal., Bioorganic & Medicinal Chemistry Letters, 2003. 13(21): p.3765-3769; each herein incorporated by reference in their entireties).

Synthesis of the Morphine-linker A is straightforward as shown in Scheme5. Morphine hydrochloric acid salt will be reacted with glutaricanhydride in basic aqueous solution to afford the desired product.

Morphine-linker B, a 3-Aromatic Acid Ester (see, e.g., Majoros, I. J.,et al., Biomacromolecules, 2006. 7(2): p. 572-579; herein incorporatedby reference in its entirety).

The 3-morphine ester of the 4-N-Boc-benzoic acid is formed in a reactionwith carbonyldiimidazole (CDI) in the presence of a base. The Boc groupis removed by treatment with 1N HCl in ether. The free amino group isimmediately reacted with glutaric anhydride to obtain themorphine-linker B (Scheme 6).

Morphine-linker C Through a Self-Immolative Linker

Self-immolative linkers have been proven to be a critical factor in thesuccess of the widely applied tripartite prodrug approach. A1,6-elimination linker (Linker A in Scheme 7), is activated by serumesterases and used to conjugate Morphine to dendrimers. Synthesis ofthis linker is well documented and its reaction with morphine is carriedout in methylene chloride in the presence of diisopropyl ethyl amine and4-(dimethylamino) pyridine (DMAP). The N-Boc group is removed under mildacidic conditions and the resulting amino group reacted with glutaricanhydride to form the desired product (Scheme 7).

Conjugation of the Morphine-Linker Compounds to the Dendrimer Attachedwith a Locking Module

Each of the Morphine-linker units are conjugated to the dendrimer whichhas already been attached with the locking module. EDC chemistry isemployed to promote the amide bond formation. The conjugates areisolated and purified using ultrafiltration and HPLC techniques.

Example 11 Conjugation of CNS Targeting Moiety

Small molecules such as folic acid (see, e.g., Majoros, I. J., et al.,Biomacromolecules, 2006. 7(2): p. 572-579; Majoros, I. J., et al.,Journal of Medicinal Chemistry, 2005. 48(19): p. 5892-5899; each hereinincorporated by reference in their entireties), peptides (RGD and

EGF) (see, e.g., Shukla, R., et al., Chemical Communications, 2005(46):p. 5739-5741; herein incorporated by reference in its entirety) and Her2antibodies (see, e.g., Shukla, R., et al., Bioconjugate Chemistry, 2006.17(5): p. 1109-1115; herein incorporated by reference in its entirety)target partially acetylated dendrimer conjugates to tumor cellsexpressing these antigens. Transferrin is used to cross the BBB and asmall peptide (Tet 1) to target neurons in the CNS. Transferrin has beenutilized as a targeting vector to transport a drugs, liposomes andproteins across the BBB by receptor mediated transcytosis (see, e.g.,Smith, M. W. and M. Gumbleton, Journal of Drug Targeting, 2006. 14(4):p. 191-214; herein incorporated by reference in its entirety).Fluorescein-conjugated, synthetic Tet1 peptide binds strongly tocultured PC12, primary motor neurons, and dorsal root ganglion (DRG)cells. Tet1 peptide also binds and internalizes in DRG and motorneurons, but not muscles in tissue sections. As such, Tet1 can be usedto target neurons once the complex reaches the CNS.

Conjugation of Transferrin

Transferrin is conjugated to the dendrimer in two steps as described inthe literature (see, e.g., Smith, M. W. and M. Gumbleton, Journal ofDrug Targeting, 2006. 14(4): p. 191-214; herein incorporated byreference in its entirety). First, a thiol reactive maleimide group isintroduced on the dendrimer by reacting with sulfo-SMCC at roomtemperature for 2 h. The dendrimer conjugate is purified by gelfiltration on a Sephadex G-25 column and subsequent dialysis. Next,sulfhydryl groups are introduced using Traut's reagent. Briefly, a coldsolution of human holotransferrin in PBS-EDTA buffer (pH 7.4) is mixedwith iminothiolane and allowed to react for 1 h. The modifiedtransferrin is purified by eluting the mixture on a PD-10 column usingPBS-EDTA buffer. The degree of thiol modification is determined usingEllman assay. The maleimide derivatized dendrimer is reacted withthiol-modified transferrin (Tf-SH) for 2 h. to give a stable thioetherbond. The reaction is stopped by quenching unreacted thiol groups withN-ethylmaleimide. The final conjugate is purified by ultrafiltration(MWCO 100,000) and analyzed by HPLC, PAGE and UV-vis spectroscopy.

Conjugation of Peptide (Tet 1)

Multiple peptide ligands on the dendrimer allow for polyvalentinteractions between the ligands and cell surface targeting molecules ina way that provides a much stronger interaction than what is achievedwith a single peptide interaction. Recently, a linear peptide (Tet 1,HLNILSTLWKYR) with the binding characteristics of tetanus toxin wasidentified by using phage display. Tet1 is used for targeted delivery ofthe narcotic analgesic/antagonist to the CNS. The peptide is synthesizedwith a terminal sulphydral linker. This peptide-SH molecule isconjugated to the dendrimer using standard protocols, and the conjugateis purified by dialysis or gel-filtration to remove excess reagents.PAGE and other analytical techniques are employed to determine theextent of peptide conjugation in the product.

Conjugation of the Morphine-Linker Compounds to the CNS TargetedDendrimer

The narcotic analgesic, Morphine, is conjugated to the dendrimer usingdifferent esterase sensitive linkers. Morphine-linker compounds can beconjugated to the dendrimer in two reaction steps. First, an activeester of the drug-linker is prepared using EDC in a DMF/DMSO solventmixture in the presence of HOBt. This active ester solution is then beadded to the aqueous solution of the dendrimer modified with a targetingand locking function. The reaction mixture is allowed to react for 24 h.(Scheme 8). The final conjugate is purified using ultrafiltration andcharacterized.

Example 12 Syntheses of Hypoxia Triggered Naloxone-Linker Units to beConjugated with CNS Targeting Dendrimer

Naloxone-indoquinone Linker (see, e.g., Naylor, M. A., et al., Journalof Medicinal Chemistry, 1997. 40(15): p. 2335-2346; Zhang, Z., et al.,Organic & Biomolecular Chemistry, 2005. 3(10): p. 1905-1910).

Synthesis of the indoquinone-Naloxone linker involves multiple steps.Starting from 2-methyl-5-methoxyindole, alkylation on nitrogen withsodium hydride and t-butyl 4-bromobutanoate, followed by formylationprovides the 3-formyl indole. After the nitration reaction, the 4-NO₂group is reduced to the amino group. Treatment with Fremy's saltproduces the indoquinone structure. The 3-formyl group is reduced andconverted to 4-NO₂-phenyl carbonate compound A. Morphine is converted tothe carbamate compound B through an activated carbonate intermediate.Compounds A and B are combined in the presence of an amine in DMF toprovide the completed drug-linker unit after an acid catalyzed t-butylester hydrolysis (Scheme 9).

Naloxone-nitroimidazole Linker (see, e.g., Hay, M. P., et al., Journalof Medicinal Chemistry, 2003. 46(25): p. 5533-5545; Hay, M. P., W. R.Wilson, and W. A. Denny, Tetrahedron, 2000. 56(4): p. 645-657; eachherein incorporated by reference in their entireties).

A nitroimidazole template is used as a reductive drug releasing systemto couple Naloxone. The one position on the drug is alkylated witht-butyl 4-bromobutanoate and the 2-hydroxymethyl group is introduced bya reaction with para-formaldehyde. After conversion of the hydroxylgroup to an activated carbonate, it is coupled with a dendrimerpreviously mentioned compound B (see Scheme 9) in the presence of abase. The t-butyl ester is removed to yield the Naloxone-linker unitthat is ready to be conjugated to the dendrimer carrier (Scheme 10).

Example 13 Design and Syntheses of Peripherally Active Drug-DendrimerConjugates Tripartite Prodrug Linker Design Strategy

The tripartite prodrug strategy has been well studied and successfullyapplied to numerous drug delivery systems especially in targeted cancertherapeutics (see, e.g., de Groot, F. M. H., E. W. P. Damen, and H. W.Scheeren, Curr. Med. Chem.—Anti-Cancer Agents, 2001. 8 p. 1093-1122;Dubowchik, G. M. and M. A. Walker, Pharmacology & Therapeutics, 1999.83: p. 67-123; Papot, S., et al., Curr. Med. Chem.—Anti-Cancer Agents,2002. 2: p. 155-185; each herein incorporated by reference in theirentireties). The general principle of drug release is shown in thefollowing simplified scheme. Namely, a trigger unit is attached to ahetero atom, such as an oxygen or nitrogen, on a unique linker, which isin turn attached to the drug of interest through a carbonate or acarbamate linkage. Once the trigger is recognized and cleaved by anenzyme in the body, the linker spontaneously decomposes to lead a facileliberation of the drug. This strategy has been proven to be effective asa powerful delivery system for various hydroxyl and amino groupcontaining drug (X═O, NH). In Scheme 11, an 1,6-elimination linker isused for illustration purposes although many other types are availableto accomplish the similar goals of drug release (see, e.g., de Groot, F.M. H., et al., Angew. Chem. Int. Ed., 2003. 42: p. 4490-4494; de Groot,F. M. H., et al., J. Org. Chem., 2001. 66: p. 8815-8830; Greenwald, R.B., et al., J. Med. Chem., 1999. 42: p. 3657-3667; Greenwald, R. B., etal., Bioconjugate Chem., 2003. 14: p. 395-403; Zhang, Z., et al.,Pharmaceutical Research, 2005. 22: p. 381-389; each herein incorporatedby reference in their entireties). As shown in Scheme 11, the dendrimerconjugates takes advantage of the serum esterase (see, e.g., Antczak,C., et al., Bioorg. & Med. Chem., 2001. 9: p. 2843-2848; Pohl, T. and H.Waldmann, J. Am. Chem. Soc., 1997. 119: p. 6702-6710; Sauerbrei, B., V.Jungmann, and H. Waldmann, Angew. Chem. Int. Ed., 1998. 37: p.1143-1146; each herein incorporated by reference in their entireties) toactivate the trigger unit in the prodrug constructs. Furthermore, theprodrug construct is conjugated to the unique dendrimer platform as adrug delivery carrier.

Synthesis of the Esterase Sensitive Linker Moieties

Typical 1,6-elimination linkers are used that can be activated by serumesterase. Since the rate of the ester bond cleavage can be significantlyaffected by the structural features at the acid side, the pivalate (see,e.g., Antczak, C., et al., Bioorg. & Med. Chem., 2001. 9: p. 2843-2848;herein incorporated by reference in its entirety) and phenylacetate(see, e.g., Pohl, T. and H. Waldmann, J. Am. Chem. Soc., 1997. 119: p.6702-6710; herein incorporated by reference in its entirety) are used asthe first set of templates to test the rate of drug release. FurtherStructure-Activity-Relationship (SAR) studies on the linkers is carriedout in order to achieve the optimal drug releasing profile.

As shown in Scheme 12, therapeutics are attached to the linker withR=t-Bu. The 5-formylsalicyclic acid is coupled withmono-Boc-ethylenediamine under an EDC catalyzed condition in DMF. Thecrude product is treated with an acyl chloride (2 eq) and triethyl amine(2.5 eq) simultaneously. After isolation and purification by silica gelchromatography, the product is reduced by boran in THF. The benzylalcohol product is treated with p-nitrobenzyl chloroformate in thepresence of pyridine and 4-(dimethylamino)pyridine (DMAP). The productis isolated and purified by silica gel chromatography.

Attachments of Ketamine and Lorazepam to the Esterase Sensitive LinkerUnits

The drugs for this study, Ketamine and Lorazepam, will be attached tothe above mentioned linkers through a displacement reaction of the4-nitrophenol group to form a carbamate linkage (Ketamine (see, e.g.,Leung, L. Y. and T. A. Baillie, J. Med. Chem., 1986. 29: p. 2396-2399;Woolf, T., et al., J. Org. Chem., 1984. 49: p. 3305-3310; each hereinincorporated by reference in their entireties)) and a carbonate linkage(Lorazepam (see, e.g., Nudelman, A., R. J. McCaully, and S. C. Bell, J.Pharm. Sci. , 1974. 63: p. 1880-1885; herein incorporated by referencein its entireties)), respectively, in the presence ofN,N-diisopropylethyl amine (DIEA) and DMAP in DMF (Scheme 13).

Conjugation of the Drug-Linker Units to the Appropriately FunctionalizedDendrimer

The partially acetylated G5 dendrimer is further modified to havecarboxylic acid at the terminal of each branch. This is achieved byreacting the partially acetylated G5 dendrimer with large excess ofglutaric anhydride. The t-Boc protecting group on the drug-linker unitis removed by treatment with 1 equivalent of 1N HCl in ether. The freedamino group is immediately conjugated with the appropriatelyfunctionalized dendrimer through an EDC catalyzed coupling reaction asshown in Scheme 14:

All the dendrimer-drug conjugates are isolated, purified, andcharacterized through methods including GPC, NMR, HPLC, UV, and CE.

Example 14 Design and Synthesis of Doxapram-Dendrimer Complex to CounterRespiratory Depression

Doxapram (Scheme 3), used for reversal of respiratory depression that isinduced by Ketamine/Lorazapram treatment, cannot be covalentlyconjugated onto dendrimer surface amines due to its structuralcharacteristics. An alternative approach is to formulate Doxapram as adendrimer complex for acid-triggered release. Dendrimer-drug complexesare formed through, for example, hydrogen bonding, hydrophobicinteractions, electrostatic interactions, or a combination of theseapproaches (see, e.g., Esfand, R. and D. A. Tomalia, Drug DiscoveryToday, 2001. 6: p. 427-436; Jansen, J. F. G. A., E. M. M. de Brabandervan den Berg, and E. W. Meijer, Science, 1994. 266: p. 1226-1229; Kolhe,P., et al., International Journal of Pharmaceutics, 2003. 259: p. 143160; Man, N., et al., European Journal of Medicinal Chemistry, 2006. 41:p. 670-674; Morgan, M. T., et al., J. Am. Chem. Soc., 2003. 125(50): p.15485-15489; Naylor, A. M., et al., J. Am. Chem. Soc., 1989. 111: p.2339-2341; Papagiannaros, A., et al., International Journal ofPharmaceutics, 2005. 302: p. 29 38; Patri, A. K., J. F.Kukowska-Latallo, and J. R. Baker, Advanced Drug Delivery Reviews, 2005.57(15): p. 2203-2214; Patri, A. K., I. J. Majoros, and J. R. Baker, Jr.,Current Opinion in Chemical Biology, 2002. 6: p. 466-471; Qiu, L. Y. andY. H. Bae, Pharmaceutical Research, 2006. 23: p. 1 30; Shcharbin, D. andB. M., Biochimica et Biophysica Acta, 2006. 1760: p. 1021-1026; eachherein incorporated by reference in their entireties). In order toprepare dendrimer-Doxapram complexes that release drug during acidosis,poly(amidoamine)

(PAMAM) dendrimers with surface succinamic acid substitutions aresynthesized to facilitate both electrostatic and hydrophobicinteractions with Doxapram.

Synthesis and Characterization of G5 Dendrimers with Varying Numbers ofSuccinamic Acid

G5 dendrimers with different succinamic acid termini are synthesized(e.g., Shi, X., et al., Electrophoresis, 2006. 27(9): p. 1758-1767;herein incorporated by reference in its entireties). Theamine-terminated G5.NH₂ dendrimers are first acetylated in differentpercentages, followed by reaction with succinic anhydride to transferthe remaining amine groups to carboxylic acid groups (Scheme 15). Theacetylation reaction (see, e.g., Majoros, I. J., et al., Macromolecules,2003. 36: p. 5526-5529; herein incorporated by reference in itsentirety), involves addition of 0.5 mL of pyridine to a 10-mL methanolsolution containing 100 mg G5.NH₂ dendrimer. Methanolic solutions (10mL) of acetic anhydride with different calculated molar ratios (25, 50,75, and 100% of the total primary amines of G5.NH₂) are added inparallel into the dendrimer/pyridine mixture solutions while vigorouslystirring, and the mixture are allowed to react for 24 h. Methanol isthen removed from the reaction mixture with a rotary evaporator. Theoily crude product is diluted with H₂O and dialyzed against water (6×4liters) for three days to remove the excess of reactants and byproducts.Water is removed from the retentate and the product is re-dissolved inwater, then lyophilized. The final G5 acetamides are annotated by usingtheir theoretical composition numbers as G5.25Ac, G5.50Ac, G5.75Ac, andG5.100Ac. For the carboxylation reaction, 50 mg dry polycationic G5acetamide derivative (acetylation percentages 0%, 25%, 50%, and 75%) isdissolved in 10 mL DMSO. Into each of the G5 acetamide solution is addedunder vigorous stirring 10 mL of DMSO solution containing succinicanhydride with 2-3 times molar excess of the remaining primary aminegroups of the particular G5 acetamide. The reaction is maintained atroom temperature for 24 h. Then, the final DMSO solution is dialyzedagainst water (6×4 liters) to remove the excess succinic anhydride aswell as the organic solvent. The aqueous retentate is filtered andlyophilized. The final G5 succinamic acids are denoted as G5.25SAH,G5.50SAH, G5.75SAH, and G5.100SAH. The synthesized dendrimer acetamidesand succinamic acids are characterized using high performance liquidchromatography (HPLC), gel permeation chromatography (GPC),matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)mass spectrometry, capillary electrophoresis (CE), and NMR (see, e.g.,Shi, X., et al., Electrophoresis, 2006. 27(9): p. 1758-1767; Islam, M.T., I. J. Majoros, and J. R. Baker, Journal of ChromatographyB-Analytical Technologies in the Biomedical and Life Sciences, 2005.822(1-2): p. 21-26; Islam, M. T., et al., Analytical Chemistry, 2005.77(7): p. 2063-2070; Shi, X., et al., Polymer, 2005. 46: p. 3022-3034;Shi, X., et al., Colloids Surf, A, 2006. 272: p. 139-150; Shi, X., I. J.Majoros, and J. R. Baker, Jr., Mol. Pharm., 2005. 2: p. 278-294; Shi, X.Y., et al., Electrophoresis, 2005. 26(15): p. 2949-2959; Shi, X. Y., etal., Analyst, 2006. 131(7): p. 842-848; Shi, X. Y., et al., Analyst,2006. 131(3): p. 374-381; Shi, X. Y., et al., Electrophoresis, 2005.26(15): p. 2960-2967; each herein incorporated by reference in theirentireties).

Formation and Characterization of G5 Dendrimer Succinamic Acid/DoxapramComplexes

Dendrimer-Doxapram complexes are prepared (see, e.g., Kolhe, P., et al.,International Journal of Pharmaceutics, 2003. 259: p. 143-160; Morgan,M. T., et al., J. Am. Chem. Soc., 2003. 125(50): p. 15485-15489; eachherein incorporated by reference in their entireties). 27 mg (8.2×10⁻⁷mol) of the G5.25SAH dendrimer (molecular weight is estimated fromprevious publication, Mw=32,910) is dissolved in 2.0 mL of CH₃OH.Doxapram hydrochloride (Mw=0.34 mg, 8.2×10⁻⁷ mol) is added to the CH₃OHsolution and stirred for 10 min. H₂O (1.0 mL) is added to the solutionand the solution is stirred for 1 h. Next, the CH₃OH is removed viaevaporation over several hours. The encapsulated drug-dendrimer solutionis then be stored at room temperature until further use. Samples for NMRanalysis are prepared using deuterated solvents. The amount of Doxapramcomplexed with G5 dendrimer succinamic acids is determined using UV-Visspectroscopy. The maximum loading capacity of Doxapram in G5 succinamicacids with different percentages of carboxyl modification is determinedAdditional methods for characterization of the complexes include NMRtechniques, zeta potential, and FTIR spectroscopy.

Example 15 Functional Analysis of Dendrimer-Opioid Conjugates andReleased Free Drug Functional Analysis of Dendrimer-Opioid Conjugatesand Released Free Drug

Binding of Morphine to a cell surface receptor initiates a series ofsignal transduction events, which lead to cellular responses. An earlyevent in the signal transduction pathway is the activation of Gi/Goproteins leading to the inhibition of adenlyate cyclase activity anddecrease in the cellular cAMP levels (see, e.g., Childers, S. R. and S.R. Childers, Opioid receptor-coupled second messenger systems. LifeSciences, 1991. 48(21): p. 1991-2003; herein incorporated by referencein its entirety). The biological functionality of the dendrimer-opioidconjugates can be determined by i) monitoring the in vitro binding ofthe conjugate onto live cells or isolated cell membranes, ii)determining the activation/inactivation of G-proteins in isolatedmembranes, and iii) quantifying the cAMP content in intact cells.

Determination of the Activation of G-Proteins Using Cell-Free MembraneSystem

The rate-limiting step in the activation of G-protein is thedissociation of bound GDP, which enables the binding of GTP at thedisplaced site. This is followed by the dissociation of G-proteinsubunits that facilitates the activation of adenlyate cyclase, and thesubsequent hydrolysis of the bound GTP by the inherent GTPase of theG-protein (see, e.g., Pierce, K. L., et al., Nature Reviews MolecularCell Biology, 2002. 3(9): p. 639-50; herein incorporated by reference inits entirety). The activity of an opioid agonist can be determined incell-free systems using partially purified membrane from opioidreceptor-expressing cells that contains the G-proteins. This is done bymeasuring the rate of agonist-stimulated membrane binding of anon-hydrolyzable analog of GTP such as the GTPγS (see, e.g., Traynor, J.R., et al., Journal of Pharmacology & Experimental Therapeutics, 2002.300(1): p. 157-61; herein incorporated by reference in its entirety), orby monitoring the membrane GTPase activity (see, e.g., Sun, H., et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica, 1995. 92(6): p. 2229-33; herein incorporated by reference inits entirety). The biochemical functionality of dendrimer-opioidconjugates is determined before and after pre-treatment with purifiedesterase by G-protein-based assays.

Evaluating Opioid Conjugate Ability to Stimulate [³⁵S] GTPγS Binding

A procedure similar to that described by Traynor et al is used (see,e.g., Traynor, J. R., et al., Journal of Pharmacology & ExperimentalTherapeutics, 2002. 300(1): p. 157-61; herein incorporated by referencein its entirety). Cells are rinsed with PBS and scraped into Tris-HClbuffer pH 7.4 containing 0.3 M sucrose in presence of proteaseinhibitors, and homogenized in a dounce homogenizer. The nuclei andunbroken cells are removed by low-speed centrifugation at 200 g and thesupernatant is spun again at 15,000 g for 20 min. The membranepreparation obtained is washed with Tris-HCl buffer and used for bindingstudies. The protein concentration of the membrane is determined usingstandard protocols. 100 μg of the partially purified membranepreparation is incubated for 10 min at 37° C. in a buffer containing[³⁵S] GTPγS and GDP. The ‘dendrimer-opioid agonist’ conjugate is thenadded and at different time points, aliquots of the reaction mixtureremoved and rapidly filtered through a glass fiber filter. The filtersare rinsed and the bound radioactivity determined by scintillationcounting. Non-specific binding is determined in presence of excess (50μM) non-radioactive GTPγS. The rate constants and maximal binding of the[³⁵S] GTPγS is determined using the GraphPad Prism program (GraphPad,San Diego, Calif.). Appropriate controls are run in the absence ofligands, and in the presence of free agonists. The effect of‘dendrimer-opioid antagonist’ on the agonist-induced binding is thenverified.

The results of the studies described above indicate if the intactdendrimer-opioid conjugates have any in vitro biological activity. The[³⁵S] GTPγS binding studies using dendrimer-Morphine conjugate that hadbeen pre-incubated with purified esterase are performed to demonstratethe biological activity of the ligand that is released by esterase. Forthis, the conjugate is incubated with commercially available esterasefor different time-periods and adding ice-cold buffer will stop theesterase action. The reaction mixture is immediately filtered through a10 kDa filter at 4° C., and the filtrate used as the ligand in the [³⁵S]GTPγS binding assay. Similarly, the filtrates obtained following hypoxictreatment of dendrimer-Naloxone conjugates are used to test whether thereleased free Naloxone inhibits agonist-induced Gi-protein activation.

Functional Determination of Opioid-Induced GTPase Activity

GTPase activity is determined (see, e.g., Sun, H., et al., Proceedingsof the National Academy of Sciences of the United States of America,1995. 92(6): p. 2229-33; herein incorporated by reference in itsentirety). Cell membranes are partially purified. 10 to 100 μg of themembrane protein are incubated in HEPES buffer pH 8.0 containingdithiothrietol and different concentrations of the ‘dendrimer-opioidagonist.’ The reaction is initiated by adding 100 nM GTP[γ-³²P] andaliquots withdrawn at different time intervals into tubes containing icecold 5% Norit A in phosphate buffer. After centrifugation of themixture, the radioactivity of the liberated [³²P]-phosphate in thesupernatant is quantified by scintillation counting. The bindingconstants are determined Appropriate controls are run in parallel, andthe effect of ‘dendrimer-opioid antagonists’ and esterase- andhypoxia-treated conjugates are also be determined If needed, thespecificity for Gi-protein activation is verified by using membranesisolated from cells that had been pre-treated with pertussis toxin (200ng/ml for 24 hrs), a treatment that inactivates the GTPase activity ofthe Gi-protein.

Determination of Opioid Mediated Cyclase Activity

Dendrimer-conjugates with either Morphine or Naloxone and in combinationare tested in vitro on opioid-responsive neuronal cell lines. OpiateMorphine binds to the mu opioid receptor (MOR), also known as theenkephalin G-protein-coupled receptor on the subsynaptic membrane ofneurons involved in the transmission of pain signals. By binding to theenkephalin receptor, Morphine enhances the analgesic effects ofenkephalin neurons. Opiate antagonists such as Naloxone bind to mureceptors, but do not activate them and prevent the binding of opiates.Morphine's relative efficacy to promote MOR internalization is much lessthan its relative efficacy to activate a G protein associated with thecytoplasmic C-terminal of the receptor (see,e.g., Borgland, S. L., etal., Journal of Biological Chemistry, 2003. 278(21): p. 18776-84; hereinincorporated by reference in its entirety). Morphine binding to areceptor triggers an allosteric change in the Ga subunit of the Gprotein, causing replacement of GDP with GTP and activation of the Gasubunit. Activated Ga, in turn, activates an effector molecule, cyclase,an enzyme in the inner side of the plasma membrane, and this enzyme thencatalyzes the conversion of ATP into the “second messenger” cyclic AMP(cAMP). Changes in the activity of Morphine and antagonism by Naloxoneof intracellular generation of cAMP will be used as a marker of thefunction of dendrimer-conjugated and released opioids. Responsiveneuronal cells in vitro are measured by cyclase ELISA (see, e.g.,Horton, J. K., et al., Journal of Immunological Methods, 1992. 155(1):p. 31-40; herein incorporated by reference in its entirety).

Example 16 Chemical Analysis of Dendrimer Conjugate and Free DrugConcentrations for Release Kinetic Studies Characterization ofGeneration 5 PAMAM Dendrimer-Drugs Conjugates

PAMAM dendrimers conjugated and/or complexed to drugs such as Ketamine,Lorazepam, Doxapram, Morphine and Naloxone are characterized. Methodsfor characterizing such conjugates have been developed (see, e.g.,Majoros, I. J., et al., Biomacromolecules, 2006. 7(2): p. 572-579;Majoros, I. J., et al., Journal of Medicinal Chemistry, 2005. 48(19): p.5892-5899; Shi, X., et al., Electrophoresis, 2006. 27(9): p. 1758-1767;Islam, M. T., I. J. Majoros, and J. R. Baker, Journal of ChromatographyB-Analytical Technologies in the Biomedical and Life Sciences, 2005.822(1-2): p. 21-26; Islam, M. T., et al., Analytical Chemistry, 2005.77(7): p. 2063-2070; Shi, X. Y., et al., Electrophoresis, 2005. 26(15):p. 2949-2959; Shi, X. Y., et al., Analyst, 2006. 131(3): p. 374-381;Shi, X. Y., et al., Electrophoresis, 2005. 26(15): p. 2960-2967; eachherein incorporated by reference in their entireties). High PerformanceLiquid Chromatography (HPLC), Size Exclusion Chromatography (SEC),Capillary electrophoresis (CE), and Matrix Assisted Laser DesorptionIonization-Time of flight (MALDI-TOF) mass spectrometric techniques areutilized.

High Performance Liquid Chromatography (HPLC)

HPLC is a widely accepted analytical method for separation andpurification of various compounds. PAMAM dendrimer and its conjugateshave been successfully characterized and analyzed using a gradient HPLCelution (see, e.g., Islam, M. T., I. J. Majoros, and J. R. Baker,Journal of Chromatography B-Analytical Technologies in the Biomedicaland Life Sciences, 2005. 822(1-2): p. 21-26; Islam, M. T., et al.,Analytical Chemistry, 2005. 77(7): p. 2063-2070; each hereinincorporated by reference in their entireties). Briefly, HPLC analysisis carried out on a Waters Delta 600 HPLC system equipped with a Waters2996 photodiode array detector, a Waters 717 Plus auto sampler, andWaters Fraction collector III. The instrument is controlled by Empower 2software. For analysis of the conjugates, a C5 silica-based RP-HPLCcolumn (250×4.6 mm, 300 Å) connected to a C5 guard column (4×3 mm) isused. The mobile phase for elution of different generations of PAMAMdendrimers is a linear gradient beginning with 100:0 (v/v)water/acetonitrile (ACN) at a flow rate of 1 mL/min. Trifluoroaceticacid (TFA) at 0.14 wt % concentration in water as well as in ACN is usedas a counter ion to make the dendrimer surfaces hydrophobic.

Capillary Electrophoresis (CE)

An Agilent Technologies CE instrument is used for this work. A proceduresimilar to as described by Shi, et. al. (see, e.g., Shi, X. Y., et al.,Electrophoresis, 2005. 26(15): p. 2949-2959; Shi, X. Y., et al.,Analyst, 2006. 131(3): p. 374-381; Shi, X. Y., et al., Electrophoresis,2005. 26(15): p. 2960-2967; each herein incorporated by reference intheir entireties) is used to characterize dendrimer conjugates. Samplesare introduced by hydrodynamic injection. Detection is done by an onlinePDA detector installed in the system.

Size Exclusion Chromatography (SEC)

SEC experiments for dendrimers and their conjugates are performed usingan Alliance Waters 2690/2695 separations module (Waters Corp., Milford,Mass.) equipped with a Waters 2487 UV absorbance detector (WatersCorp.), a Wyatt Dawn DSP laser photometer (Wyatt Technology Corp., SantaBarbara, Calif.), an Optilab DSP interferometric refractometer (WyattTechnology Corp.), and TosoHaas TSK-Gel Guard PHW 06762 (75×7.5 mm, 12μm), G 2000 PW 05761 (300×7.5 mm, 10 μm), G 3000 PW 05762 (300×7.5 mm,10 μm), and G 4000 PW (300×7.5 mm, 17 μm) columns. Citric acid buffer(0.1 M concentration) with 0.025% sodium azide in water is used as amobile phase, pH 2.74, using NaOH. Molar mass moments of the PAMAMdendrimers is determined using Astra software (version 4.9) (WyattTechnology Corp.).

MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectra is acquired using a Waters TofSpec-2Espectrometer in a reflection mode. Each sample is dissolved in 50:50mixture of methanol/water to obtain an approximate concentration of 0.25mg/mL. The samples is then mixed with equal volumes (5 μL) of the matrixsolution (10 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) dissolved inACN/ethanol (50:50)). The TFA salt form of the separated samples isisolated and collected. A 1-μL solution of the mixture is injected onthe spots of the target plate and evaporated to dryness. Calibration ofthe spectrometer is done using a mixture of known peptides in the CHCAmatrix.

NMR Spectroscopy

¹H and ¹³C and HMQC NMR spectra is taken in D₂O and used to provideintegration values for structural analysis by means of a Bruker AVANCEDRX 500 instrument. Shifts and integration of signals in the ¹H NMRspectra are used for quantitative analysis of the conjugation reactionsand for structural characterization, while the signals and shifts in the¹³C NMR spectra are used for qualitative characterization.

Example 17 Determination of In-vitro Release Kinetics of Drugs usingHPLC Ketamine, Lorazepam (in serum) and Morphine (in CSF)

HPLC is widely used in the pharmaceutical field to analyze a variety ofdrugs. Ketamine, Lorazepam and Morphine have been extensivelycharacterized using HPLC (see, e.g., Kuracka, L., et al., ClinicalChemistry, 1996. 42(5): p. 756-760; Orlovic, D., et al.,Chromatographia, 2000. 52(11/12): p. 732-734; Svensson, J., et al.,Journal of Chromatography B: Biomedical Sciences and Applications, 1982.230(2): p. 427-432; Svensson, J.-O., Journal of Chromatography B:Biomedical Sciences and Applications, 1986. 375: p. 174-178; Tebbett, I.R., Chromatographia, 1987. 23(5): p. 377-378; each herein incorporatedby reference in their entireties). These drugs are conjugated to thedendrimer scaffold using an ester linkage. The drugs are released fromthe carrier molecule into the biological matrix after cleavage bycellular esterase, tested both as purified enzyme and cellular extract.HPLC is used to qualitatively and quantitatively analyze free drugcontent in the biological medium after its release (FIG. 34).Quantitative analysis is done by a calibration curve method generated bya standard sample of the free drugs.

Sample Preparation

To study drug release using HPLC, the drug conjugated to the dendrimerscaffold is incubated with serum at 37° C. in a heparinized tube for 72hrs. Once the drug is released from the scaffold by the esterase,aliquots at various time points are collected and frozen until HPLCanalysis. Prior to HPLC analysis for free drug content, the serum sampleand cerebrospinal fluid (with and without drug conjugate) arepre-treated using the Solid Phase Extraction method carried out by usingC₁₈ Sep-pak cartridges to remove any interfering matrix impurities.After pre-treatment, the samples are reconstituted with the HPLC eluentand injected into a reverse phase column.

Quantitative Analysis—Calibrator Solutions

For quantitative analysis, standard drug samples are obtained and stocksolutions for each of these drugs are made in an appropriate solvent.Using a serial dilution method, standards for each drug at variousconcentrations are prepared to generate a calibration curve.Concentration of free drug after its release is calculated using aregression equation. Simultaneous analysis of Ketamine and Lorazepam arecarried out by preparing a sample containing both drugs. This is thenanalyzed under the specified HPLC conditions.

Recovery of the Sample (see, e.g., Kuracka, L., et al., ClinicalChemistry, 1996. 42(5): p. 756-760; Kimiskidis, V., et al., Journal ofPharmaceutical and Biomedical Analysis, (in press); each hereinincorporated by reference in their entireties)

An important step for HPLC analysis of the drug in biological medium isits extraction. The percent recovery of the sample by an extractionprocess is crucial. To check for the percent recovery of a drug samplefrom the biological medium, a standard drug sample of a knownconcentration is spiked into serum and/or cerebrospinal fluid. Thisspiked sample is then subjected to sample pre-treatment. An HPLCanalysis is performed on the pre-treated spiked sample and a standarddrug sample. From the calibration curve and the regression equation thepercent recovery is computed.

Doxapram

The synthetic approach for Doxapram involves formation of a complex withthe dendrimer, which releases the drug on lower pH (pH-trigger).Doxapram release from the dendrimer is studied in complexes formed withdifferent ratios of dendrimer and drug, and with different dendrimerswith varied amounts of succinamide substitution. The dendrimer-drugcomplex is subjected to dialysis against various buffer systems (pHranging from 7.4 to 6.8) and the dialysate subjected to HPLC analysis todetermine drug release and release kinetics. Ultrafiltration ofcomplexes mixed with serum buffered to different pH is also employed todetermine the exact drug release at varied serum pH.

Experimental Step-up

Concentrated sample of the dendrimer-Doxapram complex is prepared insaline. 500 μL of this solution is then placed in a dialysis tube(MWCO=10 KD). This sample is dialyzed against buffers of various pHranging from 6.8 to 7.4. The volume of the buffers in the outer phase is100 mL. Permeates at various time points are withdrawn from the outerphase and centrifuged against a molecular weight cut off filter toremove buffers and to concentrate the sample. The retentate isreconstituted with the eluent and subjected to analysis using HPLC. Aseries of Doxapram standards are prepared in an appropriate solventusing a serial dilution to generate a calibration curve. Quantitativeanalysis for the amount of drug release is computed using a regressionequation. In an alternative approach, to mimic physiological conditions,Doxapram release is studied in serum.

Naloxone

Release of Naloxone is triggered by hypoxia induction due to exceedinglimits of Morphine dosage. An in vitro hypoxia model that facilitatesdrug release is used for naloxone release. An in vitro hypoxia model formeasuring a neurotransmitter (Dopamine) release is used (see, e.g.,Stamford, J. A., Journal of Neuroscience Methods, 1990. 34(1-3): p.67-72; Toner, C. C. and J. A. Stamford, Journal of Neuroscience Methods,1996. 67(2): p. 133-140; Toner, C. C. and J. A. Stamford, Neuroscience,1997. 81(4): p. 999-1007; each herein incorporated by reference in theirentireties). A quantitative method for determination of drug content inartificial CSF (ACSF) using HPLC (see, e.g., Kimiskidis, V., et al.,Journal of Pharmaceutical and Biomedical Analysis, (in press); hereinincorporated by reference in its entirety). Also, Naloxone has beenthoroughly characterized and analyzed using HPLC (see, e.g., Achilli,G., et al., Journal of Chromatography A, 1996. 729(1-2): p. 273-277;herein incorporated by reference in its entirety). Accordingly, an invitro model using HPLC to study the CSF release kinetics of Naloxoneunder hypoxic conditions is provided.

Experimental Design

An in vitro hypoxia model is provided. The sample vials are placed in anenclosed chamber that has the capability to continuously allow the flowof required gases and alternatively facilitates ease of sampling. Theamount of gas that pumped into this chamber is maintained and controlledthrough a peristaltic pump. The temperature of the system is maintainedat 37° C. Using this set-up, the sample vials containing dendrimer-drugconjugate in CSF are incubated in this enclosed chamber. Varyingconcentrations of human DT-diaphorase (Sigma), the brain enzymeresponsible for the bioreduction catalysis of the indolequinone-Naloxoneconjugate that is regulated by hypoxia, are provided. Initially, hypoxiais induced by gassing the chamber with 95% N₂/5% CO₂ gas for about 30minutes at 400 ml/hr (see, e.g., Toner, C. C. and J. A. Stamford,Neuroscience, 1997. 81(4): p. 999-1007; herein incorporated by referencein its entirety). Simultaneously, another sample vial is oxygenated byflowing 95% O₂/5%CO₂ that serves as a control. After sample incubationunder hypoxic condition, aliquots from each sample (oxygenated anddeoxygenated) are taken and stored until analysis. For sample incubatedunder hypoxic conditions, storage of the aliquots, sample pre-treatmentand sample preparation are carried out in a glove box to maintainhypoxic conditions. HPLC of these samples is then carried out to checkfor free drug content.

Drug Release Kinetics

The sample is incubated under 95% N₂/5% CO₂ atmosphere for differenttime periods and aliquots are collected at the end of each incubationtime period to study the amount of drug release. Also, the rate ofgassing (the amount of gas passing through the chamber) is critical. Tooptimize these conditions, the chamber is gassed at various speeds andat the end of each run, the aliquots are taken for analysis using HPLC.Once the conditions for hypoxia induced drug release are optimized, thesamples in CSF are subjected to the set conditions to characterize thekinetics of drug release. The samples are pre-treated prior to HPLCanalysis. Complete quantitative analysis using standard samples ofNaloxone is carried out in a similar fashion.

Example 18 Apparatus and Chromatographic Conditions to Study ReleaseKinetics of Drugs

The complete qualitative and quantitative analysis using HPLC is carriedout on a Waters Delta 600 HPLC system equipped with a Waters 2996photodiode array detector, a Waters 717 Plus auto sampler, WatersFraction collector III and Empower 2 software. An analytical size column(C8 or C18) with a particle size of 5 μ is used. Initially, an isocraticelution using acetonitrile/phosphate buffer, pH 7.0, is used. Theconditions for an HPLC experiment, if required, are modified in order toincorporate various analyses and generate efficient results.

Example 19

Synthesized analgesic nanodevices should be stable and biologicallyinactive. The biological activity (or lack thereof) is tested using theradio-ligand competition-binding assay. If no binding competition isobserved, the nanodevices are treated to release fully functional drugs.The drugs' release kinetics and biological activity is further evaluatedusing fluorescence polarization immunoassay (FPIA) analysis and theradioligand competition-binding assay. This assay is used to study theability of released drug and drug conjugates to bind to the respectivereceptors.

The assay is performed using membrane preparations from cell lines,shown in Table 2, that express various receptors. Prior to membranepreparation, the cells are maintained in RPMI 1640 medium supplementedwith 10% heat-inactivated bovine calf serum, 2mM L-glutamine, as well aspenicillin and streptomycin. The cells are grown at 37° C. in a 5% CO₂incubator. Cell membranes are prepared as described previously (see,e.g., Homer, K. A., et al., Brain Research, 2004. 1028(2): p. 121-32;herein incorporated by reference in its entirety). Total proteinconcentration is determined with bovine serum albumin (BSA) as astandard. Cell membrane binding assays for receptors are performed asdescribed previously (see, e.g., Homer, K. A., et al., Brain Research,2004. 1028(2): p. 121-32; herein incorporated by reference in itsentirety). Briefly, cell membranes (0.1 mg protein) are incubated in 100mM Trizma/0.3% bovine serum albumin containing a constant concentrationof ³{tilde over (H)} drug and various concentrations of unlabeled drugeither coupled to or released from the dendrimer. Nonspecific binding isdefined as that measured in the presence of 1 μM of cold drug. Themembranes are filtered onto Skatron glass fiber filters that have beensoaked in 50 mM Trizma, using a Skatron harvester (Molecular Devices,Sunnyvale, Calif.). Filter disks are placed in scintillation cocktail(Ready-Protein Plus, Beckman Coulter, Fullerton, Calif.) and counted.Total binding is defined as dpm of ³{tilde over (H)} drug drug bound byeach sample. Each concentration of drug is assayed in triplicate and theexperiment repeated at least 2 times. Nonlinear regression analysis of³{tilde over (H)}drug competition assays is performed with GraphPadPrism (GraphPad Software, San Diego, Calif., USA). ³H—Ketamine and³H—Doxapram is radio-labeled as described previously (see, e.g., Adams,J. D., Jr., et al., Biomedical Mass Spectrometry, 1981. 8(11): p.527-38; herein incorporated by reference in its entirety).

Receptors/ Cell line Drugs transporters Cell lines availabilityReferences Morphine μ opioid SH-SY5Y, From ATCC Horner, K. A., et al.,Brain receptor SK-N-SH, Research, 2004. 1028(2): T47D p. 121-32; hereinincorporated by reference in its entirety Naloxone μ opioid SH-SY5Y,From ATCC Horner, K. A., et al., Brain receptor SK-N-SH, Research, 2004.1028(2): T47D p. 121-32; herein incorporated by reference in itsentirety Doxapram CO₂ PC12 From ATCC Millhorn, D. E., et al.,chemoreceptor Advances in Experimental Medicine & Biology, 1996. 410: p.135-42; herein incorporated by reference in its entirety KetamineN-methyl-D- NG108-15 From ATCC Cai, Y. C., et al., aspartate MolecularPharmacology, receptor 1997. 51(4): p. 583-7; herein incorporated byreference in its entirety Lorazepam glutamate PC-3 From ATCC Franklin,R. B., et al., transporter BMC Biochemistry, 2006. EAAC1 7: p. 10;herein incorporated by reference in its entirety

Table 2. shows drugs, receptors and cell lines expressing thesereceptors.

Fluorescence Polarization Immunoassay (FPIA) Analysis

To evaluate of kinetics of Morphine and Naloxone release from thenanodevice, the fluorescence polarization immunoassay is performed asdescribed previously (see, e.g., KuKanich, B., et al., Therapeutic DrugMonitoring, 2005. 27(3): p. 389-92; herein incorporated by reference inits entirety). FPIA samples are prepared by mixing of 100 _([)μLstandard solution or supernatant of drug filtered through a 10 kDacutoff membrane to separate drug released from nanodevice. The materialsare mixed with 100 μL of the tracer solution in borate buffer and 300 μLof optimal dilution of MAb. The reaction mixture is vortexed andfluorescence polarization will be measured using a Beacon 2000instrument with a variable temperature unit (Pan Vera Corp., Madison,Wis.). Standard curves are obtained by plotting normalized fluorescencepolarization signal (100×B/B₀, where B₀=fluorescence polarization of“zero” standard, B=fluorescence polarization of each standard or resteddrug solution) against the logarithm of the analyte concentration.Sigmoidal curves are fitted to a four-parameter logistic equation.

Example 20 Testing the Movement of Dendrimer-Drug Conjugates Across anArtificial BBB

To demonstrate that the CNS targeted dendrimer conjugate is transportedacross the BBB, the DIV-BBB system (Flocel Inc., Cleveland, Ohio) thatclosely mimics the in vivo BBB is used (see, e.g., Cucullo, L., et al.,Current Opinion in Drug Discovery & Development, 2005. 8 (1): p. 88-99;herein incorporated by reference in its entirety). Briefly, the DIV-BBBis characterized by tight junctions, segregated lumenal/ablumenaltransporters (i.e. potassium, amino acids, glucose GLUT-1), a negligiblepermeability to 14C sucrose, and a high transendothelial electricalresistance (TEER>1000 Ωcm2). The dendrimer-drug conjugate is dissolvedin saline and injected through the luminal side of the DIV-BBBcartridge. The conjugate is allowed to pass through the endothelialcapillaries (EC) to the ablumenal side. The samples are then becollected from the ablumenal side at various time intervals and analyzedfor the presence of conjugate using HPLC.

Evaluation of Locking Module

Dendrimers with varying degrees of substitution with thetetrahydronicotinamide derivative are exposed to horseradish peroxidaseenzymes (Sigma) in varying buffers, neuronal cell lines, enzyme andprotein concentrations to simulate the CNS environment. MS and protonNMR determine the amount of charged material on the dendrimer. Thismaterial is tested for the ability to cross the BBB. Transport beforeand after treatment is compared, as is activity with different levels ofconjugate to determine an optimal degree of conjugation to developinducible locking activity.

Neuronal Cell Binding Assays

The ability of transferrin and Tet1 dendrimer conjugates to bind toneurons is evaluated using several techniques. Neuronal cell lines(SH-SY5Y, SK-N-SH, T47D, see Table 2 above) are cultured and incubatedwith radio-labeled transferrin and Tet1 dendrimer conjugates. Cells arewashed and the cell pellets collected and assayed for boundradioactivity. Non-labeled transferrin and tet peptides are employed inan effort to block binding. Conjugates having both drug and targetingligand are employed to determine if there is cooperative binding.Alternatively, the dendrimer conjugates are labeled with fluorescein andthe binding and uptake in the neuronal cells tested using confocalmicroscopy and flow cytometry (see, e.g., Majoros, I. J., et al.,Biomacromolecules, 2006. 7(2): p. 572-579; Shukla, R., et al.,Bioconjugate Chemistry, 2006. 17(5): p. 1109-1115; each hereinincorporated by reference in their entireties). These techniques allowfor rapid “dose ranging” studies.

Example 21 Morphine and Naloxone Pro-Drugs, Dendrimer-Pro-Drug Complexesand Dendrimer-Pro-Drug Conjugates Synthesized

Morphine and Naloxone were first modified to form pro-drugs through theattachment of rationally designed chemical modifications (FIGS. 35, 36,and 37). The modifications used for the Morphine pro-drugs were designedto be progressively and consistently cleaved by the esterases present inplasma, while those used in the formation of the Naloxone pro-drugs werecleaved to yield active drug by reduction only under hypoxic conditions,therefore serving as the feedback mechanism. Structural variations inthe linkers resulted in variations in the rate of the continuous releaseof Morphine and the hypoxia-activated release of Naloxone. Both sets ofpro-drugs were then associated with G5 PAMAM dendrimers to form adelivery platform through either i) non-covalent complexation with thepolymer or ii) covalent conjugation to the dendrimer. A schematicillustrating the family of resulting formulations is in vivo displayedin FIG. 38.

Morphine Pro-Drug Complexes and Dendrimer Morphine Pro-Drug Conjugatescan be used to Obtain a Release of 2.25 mg Morphine/Hour in HumanPlasma.

Release rates of Morphine from Morphine-pro-drug, dendrimer Morphinepro-drug complexes and dendrimer-Morphine pro-drug conjugates wereobtained. These studies were completed by incubating each compound withhuman plasma for a given period of time. Following incubation withplasma, the release of Morphine from the Morphine compounds wasquantified using HPLC. The identities of the HPLC peaks were confirmedusing mass spectrometry analysis and the amount of Morphine released wascalculated using a calibration curve. FIG. 39 shows the time-dependentrelease of Morphine from the different Morphine compounds afterincubation with human plasma. These formulations were used incombination to achieve 2.25 mg Morphine/hour and demonstrate thefeasibility of achieving the controlled release of Morphine.

Naloxone Pro-Drug can be used to Obtain a Release of 6 mg Naloxone/Hourin Human Plasma Under Hypoxic Conditions (pO₂ of 18 mmHg).

The release rate of Naloxone from an indolequinone linker-based Naloxonepro-drug was obtained. This study was completed by incubating theNaloxone pro-drug with human plasma for a given period of time at a pO₂of 18 mmHg Following this incubation, the release of Naloxone from theNaloxone pro-drug was quantified using HPLC. The identities of the HPLCpeaks were confirmed using mass spectrometry analysis and the amount ofNaloxone released was calculated using a calibration curve. FIG. 40shows the time-dependent release of Naloxone from the Naloxone pro-drugafter incubation with human plasma. Based on this release study, it wasdetermined that 78 mg of the indolequinone linker-based Naloxonepro-drug are required to achieve a release of 6 mg Naloxone/hour.

Example 22 Scale-up of Synthesized Morphine/Hydro-Morphone and NaloxoneCompounds

In order to provide effective narcotic (Morphine/Hydromorphone)analgesia over prolonged periods, scale-up synthesis processes for thecompounds are developed. Three (3) grams of each compound aresynthesized. These compounds are used in combination to obtain thedesired release kinetics of the analgesic and narcotic antagonist.

Synthesis of Morphine/Hypdromorphone Pro-Drugs.

All of the Morphine pro-drugs (A, B, C, D, E, F, G; see FIG. 36) weresynthesized through conventional organic chemistry (FIG. 35). Yields foreach step were moderate to excellent. To minimize the number ofpermeatations, four of the six Morphine pro-drugs synthesized areselected for scale-up. Given the increased potency of Hydromorphone ascompared to Morphine, two esterase-s7ensitive Hydromorphone pro-drugsare sythesized on gram scales.

Synthesis of Naloxone Pro-Drugs.

The synthesis of indolequinone linker-based Naloxone pro-drugs wasachieved on the 10-20 mg scale. Although 8 indolequinone-based Naloxonepro-drugs were originally synthesized, only the 4 pro-drugs containingdiamine spacers were shown to be stable in PBS buffer. Therefore, onlythese 4 pro-drugs are scaled up to gram quantities (FIG. 36). The finalindolequinone-Naloxone products are further purified by preparation TLC(0.25 mm thick, 20×20 cm, Whatman).

Synthesis of Dendrimer-Pro-Drug Complexes.

An advantage of using dendrimer/drug complexes for drug deliveryapplications is generally known (1) to make water-insoluble drugs watersoluble, and (2) to achieve high drug loading capacity. The hydrophobicinterior of dendrimers along with their unique surface functional groupsallow for effective complexation of drugs (FIG. 41). The mechanism toform dendrimer/drug complexes is based on the hydrophobic interaction,electrostatic interaction, hydrogen bonding, Van der Waals force, and/orthe combination thereof (see, e.g., Esfand, R. and D. A. Tomalia, DrugDiscovery Today, 2001. 6: p. 427-436; Kolhe, P., et al., InternationalJournal of Pharmaceutics, 2003. 259: p. 143-160; Morgan, M. T., et al.,J. Am. Chem. Soc., 2003. 125(50): p. 15485-15489; Papagiannaros, A., etal., International Journal of Pharmaceutics, 2005. 302: p. 29-38; Patri,A. K., J. F. Kukowska-Latallo, and J. R. Baker, Jr., Advanced DrugDelivery Reviews, 2005. 57: p. 2203-2214; Shcharbin, D. and B. M.,Biochimica et Biophysica Acta, 2006. 1760: p. 1021-1026; each hereinincorporated by reference in their entireties. In experiments conductedduring the course of the present invention it was shown that Morphineand Naloxone pro-drugs can be complexed with G5 dendrimers with veryhigh payload (40-70 pro-drug molecules/per dendrimer molecule). Thenovel concept to use dendrimer/pro-drug complexes allows one to designdesired bimodal release profiles of drugs through different mechanisms.For example, a dendrimer/Morphine pro-drug complex can be first releasedthrough a general diffusion-driven mechanism, followed by the cleavageof the ester bond of the pro-drugs to release Morphine drug.

To obtain gram scales of the Morphine/Hydromorphone or Naloxone pro-drugcomplexes, a G5 dendrimer with 80 amine groups acetylated and theremaining amines carboxylated (G5.NHAc80-SAH) is used for complexation.G5.NHAc₈₀-SAH dendrimer (1.0 g) is dissolved in 50-mL water, and aspecific pro-drug (molar ratio of dendrimer/pro-drug=1:80) dissolvedinto 5 mL methanol. The two solutions are mixed together andmagnetically stirred over night to allow the evaporation of methanol.Then, the solution is centrifuged to remove possible precipitatesrelated to non-complexed Morphine or Naloxone pro-drugs as the pro-drugsare hydrophobic and insoluble in water. The precipitate is collected anddissolved into methanol for HPLC analysis. The supernatant islyophilized. The loading capacity of drugs is determined using HPLC. Thefinal complexes are characterized using NMR, zeta potentialmeasurements, and light scattering.

Synthesis of Dendrimer Pro-Drug Conjugates.

In experiments conducted during the course of the present invention,several dendrimer pro-drug conjugates were synthesized. The pro-drugscontaining acid, amine or azide functional groups were reacted withcarboxyl, amine, hydroxyl or alkyne surface modified dendrimers,respectively, to afford the desired dendrimer drug compounds (FIG. 42).A similar strategy is utilized to scale up these compounds (100 mg-1 g).In experiments conducted during the course of the present invention, itwas shown that the synthesis of folic acid targeted dendrimer-drugconjugates can be scaled up to 100 g without any significantpurification problems. A G5 PAMAM dendrimer is used as a generalplatform. G5 PAMAM dendrimer is first partially acetylated using acalculated amount acetic anhydride in presence of triethylamine as base.The remaining amino groups of the dendrimer are further modified to haveeither carboxylic acid or alkyne groups at the terminus of each branch.The carboxyl surface modification is achieved by reaction of thepartially acetylated G5 dendrimer with a large excess ofsuccinic/glutaric anhydride in methanol. The partially acetylated G5dendrimer is reacted with 3-(4-(ethynyloxy)phenyl)propanoic acid to givealkyne functionalized partially acetylated dendrimer. Pro-drugscontaining a carboxyl functional group are reacted with partiallyacetylated dendrimer to provide amide linked dendrimer-drug conjugate.The carboxyl surface modified dendrimer is attached to the pro-drugshaving amine functional groups using standard carbodiimide couplingreaction. In experiments conducted during the course of the presentinvention, a method was developed for reacting azide functionalized druglinkers with alkyne terminated dendrimers using Cu(I) as catalyst. Allthese conjugate syntheses are readily scaled-up to gram quantities usinga variety of coupling conditions. The synthesized conjugates arepurified by gel filtration, large scale ultrafiltration using membranesof large surface area and dialysis, and characterized using NMR, massspectroscopy and UV-vis.

Chemical Analysis of Dendrimer Complexes/Conjugates and Free DrugConcentration for Release Kinetic Studies. Characterization ofGeneration 5 PAMAM Dendrimer-Drugs Conjugates.

In experiments conducted during the course of the present invention,successful methods were developed to characterize PAMAM dendrimers (see,e.g., Majoros, I. J., et al., Biomacromolecules, 2006. 7(2): p. 572-579;Majoros, I. J., et al., Journal of Medicinal Chemistry, 2005. 48(19): p.5892-5899; Islam, M. T., I. J. Majoros, and J. R. Baker, Journal ofChromatography B-Analytical Technologies in the Biomedical and LifeSciences, 2005. 822(1-2): p. 21-26; Islam, M. T., et al., AnalyticalChemistry, 2005. 77(7): p. 2063-2070; Shi, X., et al., Electrophoresis,2006. 27(9): p. 1758-1767; Shi, X. Y., et al., Electrophoresis, 2005.26(15): p. 2949-2959; Shi, X. Y., et al., Analyst, 2006. 131(3): p.374-381; Shi, X. Y., et al., Electrophoresis, 2005. 26(15): p.2960-2967; each herein incorporated by reference in their entireties).High Performance Liquid Chromatography (HPLC), Size ExclusionChromatography (SEC), Capillary electrophoresis (CE), and MatrixAssisted Laser Desorption Ionization-Time of flight (MALDI-TOF) massspectrometric techniques are utilized.

High Performance Liquid Chromatography (HPLC)

HPLC is a widely accepted analytical method for separation andpurification of various compounds. PAMAM dendrimer and its conjugateshave been successfully characterized and analyzed using a gradient HPLCelution (see, e.g., Islam, M. T., I. J. Majoros, and J. R. Baker,Journal of Chromatography B-Analytical Technologies in the Biomedicaland Life Sciences, 2005. 822(1-2): p. 21-26; Islam, M. T., et al.,Analytical Chemistry, 2005. 77(7): p. 2063-2070; each hereinincorporated by reference in their entireties). Briefly, HPLC analysisis carried out on a Waters Delta 600 HPLC system equipped with a Waters2996 photodiode array detector, a Waters 717 Plus auto sampler, andWaters Fraction collector III. The instrument is controlled by Empower 2software. For analysis of the conjugates, a C5 silica-based RP-HPLCcolumn (250×4.6 mm, 300 Å) connected to a C5 guard column (4×3 mm) isused. The mobile phase for elution of different generations of PAMAMdendrimers is a linear gradient beginning with 100:0 (v/v)water/acetonitrile (ACN) at a flow rate of 1 mL/min. Trifluoroaceticacid (TFA) at 0.14 wt % concentration in water as well as in ACN areused as a counter ion to make the dendrimer surfaces hydrophobic.

Capillary Electrophoresis (CE)

An Agilent Technologies CE instrument is used for capillaryelectrophoresis. A procedure is used to characterize dendrimerconjugates (see, e.g., Shi, X. Y., et al., Electrophoresis, 2005.26(15): p. 2949-2959; Shi, X. Y., et al., Analyst, 2006. 131(3): p.374-381; Shi, X. Y., et al., Electrophoresis, 2005. 26(15): p.2960-2967; each herein incorporated by reference in their entireties).Samples are introduced by hydrodynamic injection. Detection is done byan online PDA detector installed in the system.

Size Exclusion Chromatography (SEC)

SEC experiments for dendrimers and their conjugates are performed usingan Alliance Waters 2690/2695 separations module (Waters Corp., Milford,Mass.) equipped with a Waters 2487 UV absorbance detector (WatersCorp.), a Wyatt Dawn DSP laser photometer (Wyatt Technology Corp., SantaBarbara, Calif.), an Optilab DSP interferometric refractometer (WyattTechnology Corp.), and TosoHaas TSK-Gel Guard PHW 06762 (75×7.5 mm, 12μm), G 2000 PW 05761 (300×7.5 mm, 10 μm), G 3000 PW 05762 (300×7.5 mm,10 μm), and G 4000 PW (300×7.5 mm, 17 μm) columns. Citric acid buffer(0.1 M concentration) with 0.025% sodium azide in water is used as amobile phase, pH 2.74, using NaOH. Molar mass moments of the PAMAMdendrimers are determined using Astra software (version 4.9) (WyattTechnology Corp.).

MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectra is acquired using a Waters TofSpec-2Espectrometer in a reflection mode. Each sample is dissolved in a 50:50mixture of methanol/water to obtain an approximate concentration of 0.25mg/mL. The samples are then mixed with equal volumes (5 μL) of thematrix solution (10 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA)dissolved in ACN/ethanol (50:50)). The TFA salt form of the separatedsamples is isolated and collected. A 1-μL solution of the mixture isinjected on the spots of the target plate and evaporated to dryness.Calibration of the spectrometer is done using a mixture of knownpeptides in the CHCA matrix.

NMR Spectroscopy

¹H and ¹³C and HMQC NMR spectra is taken in D₂O and used to provideintegration values for structural analysis by means of a Bruker AVANCEDRX 500 instrument. Shifts and integration of signals in the ¹H NMRspectra are used for quantitative analysis of the conjugation reactionsand for structural characterization, while the signals and shifts in the¹³C NMR spectra are used for qualitative characterization.

Example 23 Perform in vitro Binding and Cytotoxicity Studies ofMorphine/Hydromorphone and Naloxone Compounds

The controlled drug release is based on the premise that the synthesizedpro-drugs remain biologically minimally active or inactive and the freedrug is released to an active form by a physiological trigger such as anesterase action or hypoxia. The biological function of the pro-drugs andthe released drugs by bioassays are monitored. The bindingcharacteristics of the released drugs on appropriate cells which expressthe receptors for these drugs are tested. Ligands with establishedbiochemical signal effects following their binding are studied bymonitoring these signals. For example, the binding of Morphine to a cellsurface receptor initiates a series of signal transduction events, whichlead to several cellular responses. An early event in the signaltransduction pathway is the activation of Gi/Go proteins leading to theinhibition of adenlyate cyclase activity and decrease in the cellularcAMP levels (see, e.g., Childers, S. R. and S. R. Childers, LifeSciences, 1991. 48(21): p. 1991-2003; herein incorporated by referencein its entirety). The biological functionality of the dendrimer-opioidconjugates are determined in vitro by, for example, i) monitoring thebinding of the conjugate onto live cells or isolated cell membranes, ii)determining the activation/inactivation of G-proteins in isolatedmembranes, and iii) quantifying the cAMP content in intact cells.

Radio-Ligand Competition-Binding Assay

This assay is performed using membrane preparations from cell lines,shown in Table 3, that express various receptors.

TABLE 3 Analgesic drugs, receptors and cell lines expressing thesereceptors. Receptors/ Cell line Drugs transporters Cell linesavailability References Morphine/ μ opioid SH-SY5Y, From ATCC Horner, K.A., et al., Brain Hydromorphone receptor SK-N-SH, Research, 2004.1028(2): p. T47D 121-32; herein incorporate by reference in its entiretyNaloxone μ opioid SH-SY5Y, From ATCC Horner, K. A., et al., Brainreceptor SK-N-SH, Research, 2004. 1028(2): p. T47D 121-32; hereinincorporate by reference in its entiretyCells are maintained in appropriate culture medium supplemented with 10%heat-inactivated bovine calf serum, 2 mM L-glutamine, as well aspenicillin and streptomycin, and grown at 37° C. in a 5% CO₂ incubator.Cell membranes are prepared as described previously (see, e.g., Homer,K. A., et al., Brain Research, 2004. 1028(2): p. 121-32; hereinincorporate by reference in its entirety). Total protein concentrationis determined and membrane binding assays for receptors are performed asdescribed previously (see, e.g., Homer, K. A., et al., Brain Research,2004. 1028(2): p. 121-32; herein incorporate by reference in itsentirety). Briefly, cell membranes (0.1 mg protein) are incubated in 50mM Tris-HCl pH 7.4/0.3% bovine serum albumin containing a constantconcentration of ³H-drug and various concentrations of unlabeled drugeither coupled to or released from the dendrimer. Nonspecific binding isdefined as that measured in the presence of 1 μM of cold drug. Themembranes are then filtered onto Skatron glass fiber filters that havebeen soaked in 50 mM Tris-HCl, pH 7.4, using a Skatron harvester(Molecular Devices, Sunnyvale, Calif.). Filter disks are placed inscintillation cocktail (Ready-Protein Plus, Beckman Coulter, Fullerton,Calif.) and counted. Total binding is defined as dpm of ³H drug bound byeach sample. Each concentration of drug is assayed in triplicate and theexperiment repeated at least two times. Nonlinear regression analysis of³H-drug competition assays is performed with GraphPad Prism (GraphPadSoftware, San Diego, Calif., USA).

Determination of Receptor Binding of Drug Compounds by Surface PlasmonResonance (SPR).

The binding of the synthesized pro-drugs and dendrimer-drug compoundsonto receptors of these drugs in partially purified membrane fractionsof cell lines listed in Table 3 is tested. For this, membrane isextracted with 0.5% Triton-X in buffer containing protease inhibitorsand centrifuged at 100,000×g for 1 h. The supernatant is ultra filteredto remove the detergent and other small molecules, and the proteinextract used as a source of receptors for drug-conjugate binding. Ifneeded, the opioid receptor is further purified by affinitychromatography of the Triton-X-solubilized fraction. The binding of theconjugates is tested using a BIAcore X instrument (BIAcore AB, Uppsala,Sweden) (see, e.g., Hong, S., et al., Chemistry & Biology, 2007. 14(1):p. 105-113; herein incorporated by reference in its entirety). Theconditions for receptor immobilization and ligand binding is optimizedusing different BIAcore sensor chips and buffer solutions as suggestedby the vendor. The data obtained is analyzed by a global fitting bindingmodel using the BIAevaluation 3.2 software. The apparent equilibriumdissociation constants (K_(a)) are calculated from the ratio of thedissociation and association rate constants (k_(off)/k_(on)). The dataobtained displays the efficacy for binding of the conjugates vs. freedrugs on their respective receptors.

Determination of the Opioid Receptor Activation.

The rate-limiting step in the activation of G-protein is thedissociation of bound GDP, which enables the binding of GTP at thedisplaced site. This is followed by the dissociation of G-proteinsubunits that facilitates the activation of adenlyate cyclase and thesubsequent hydrolysis of the bound GTP by the inherent GTPase of theG-protein (see, e.g., Pierce, K. L., et al., Nature Reviews MolecularCell Biology, 2002. 3(9): p. 639-50; herein incorporated by reference inits entirety). The activity of an opioid agonist is determined incell-free systems using partially purified membrane from opioidreceptor-expressing cells that contains the G-proteins. This is done bymeasuring the rate of agonist-stimulated membrane binding of anon-hydrolyzable analog of GTP such as the GTPγS (see, e.g., Traynor, J.R., et al., Journal of Pharmacology & Experimental Therapeutics, 2002.300(1): p. 157-61; herein incorporated by reference in its entirety), orby monitoring the membrane GTPase activity (see, e.g., Sun, H., et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica, 1995. 92(6): p. 2229-33; herein incorporated by reference inits entirety). The biochemical functionality of dendrimer-opioidconjugates before and after pre-treatment with purified enzymes andplasma is determined by G-protein-based assays.

Evaluating Opioid Conjugate Ability to Stimulate [³⁵S] GTPγS Binding.

Cells are rinsed with PBS and scraped into 50 mM Tris-HCl buffer pH 7.4containing 0.3 M sucrose in presence of protease inhibitors, andhomogenized in a dounce homogenizer. The nuclei and unbroken cells areremoved by low-speed centrifugation at 200×g and the supernatant is spunagain at 15,000×g for 20 min. The membrane preparation obtained iswashed with Tris-HCl buffer and used for binding studies. The proteinconcentration of the membrane is determined using standard protocols.100 μg of the partially purified membrane preparation is incubated for10 min at 37° C. in a buffer containing [³⁵S] GTPγS and GDP. Thepro-drugs are then added and at different time points, aliquots of thereaction mixture removed and rapidly filtered through a glass fiberfilter. The filters are rinsed and the bound radioactivity determined byscintillation counting. Non-specific binding is determined in presenceof excess (50 μM) non-radioactive GTPγS. The rate constants and maximalbinding of the [³⁵S] GTPγS is determined using the GraphPad Prismprogram (GraphPad, San Diego, Calif.). Appropriate controls are run inthe absence of ligands, and in the presence of free agonists. The effectof the ‘dendrimer-opioid antagonist’ on the agonist-induced binding isthen be verified.

The results of the studies described above indicate if the intactpro-drugs have any in vitro biological activity. The [³⁵S] GTPγS bindingstudies using pro-drugs that had been pre-incubated with purifiedesterase/plasma are done in order to demonstrate the biological activityof the ligand that is released by esterase. For this, theconjugate/complex is incubated with the enzymes for differenttime-periods and adding ice-cold buffer to stop the esterase action. Thereaction mixture is immediately filtered through a 10 kDa filter at 4°C., and the filtrate used as the ligand in the [³⁵S] GTPγS binding assay(see, e.g., Traynor, J. R., et al., Journal of Pharmacology &Experimental Therapeutics, 2002. 300(1): p. 157-61; herein incorporatedby reference in its entirety). Similarly, the filtrates obtainedfollowing hypoxic treatment of dendrimer-Naloxone pro-drugcomplexes/conjugates are used to test whether the released free Naloxoneinhibits agonist-induced Gi-protein activation.

Functional Determination of Opioid-Induced GTPase Activity.

GTPase activity will be determined as previously described (see, e.g.,Sun, H., et al., Proceedings of the National Academy of Sciences of theUnited States of America, 1995. 92(6): p. 2229-33; herein incorporatedby reference in its entirety). Cell membranes are partially purified. 10to 100 μg of the membrane protein are incubated in HEPES buffer pH 8.0containing dithiothrietol and different concentrations of pro-drugs. Thereaction is initiated by adding 100 nM GTP[γ-³²P] and aliquots withdrawnat different time intervals into tubes containing ice cold 5% Norit A inphosphate buffer. After centrifugation of the mixture, the radioactivityof the liberated [³²P]-phosphate in the supernatant is quantified byscintillation counting. The binding constants are determined Appropriatecontrols are run in parallel, and the effect of respiratory stimulantsand esterase- and hypoxia-treated conjugates are also determined. Ifneeded, the specificity for Gi-protein activation is verified by usingmembranes isolated from cells that had been pre-treated with pertussistoxin (200 ng/ml for 24 hrs), a treatment that inactivates the GTPaseactivity of the Gi-protein.

Determination of Adenylate Cyclase Activity.

Dendrimer-based pro-drugs with either Morphine/Hydromorphone or Naloxoneand in combination are tested in vitro on opioid-responsive neuronalcell lines. Morphine binding and G-protein mediated signal transductionevents leads to the activation of the enzyme adenlyate cyclase andgeneration of the second messenger cyclic AMP (cAMP). Changes in theactivity of Morphine and antagonism by Naloxone of intracellulargeneration of cAMP are used as a marker of the function ofdendrimer-conjugated and released opioids. Responsive neuronal cells aremeasured by cyclase ELISA (see, e.g., Horton, J. K., et al., Journal ofImmunological Methods, 1992. 155(1): p. 31-40; herein incorporated byreference in its entirety).

In vitro Cytotoxicity Studies.

The cytotoxicity of the free drugs and the conjugates is determined bymultiple biochemical assays such as XTT assay, Clonogenic assay andlactate dehydrogenase (LDH) release assay, using the cells lines givenin Table 3. As some indolequinones have variable cytotoxic potential inthe oxidized and reduced state (see, e.g., Newsome et al, Organic &Biomolecular Chemistry 5(10): 1629-40, 2007; herein incorporated byreference in its entirety), the cytotoxicity of the compounds before andafter reduction by reductases is tested.

XTT assay.

This assay is based on the conversion of XTT (sodium3′-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid hydrate; Roche Diagnostics) to formazan bymitochondria of live cells. Cells are placed in 96-well microtiterplates and incubated with various concentrations of the drug conjugatesfor different time periods. The drugs are removed and the cells areallowed to recover for 3 days. The cells are incubated with XTT reagentfor 2 hours and the absorbance of the formazan product formed ismeasured at 492 nm on ELISA reader, using a reference wavelength of 690nm.

Clonogenic Assay.

This is based on inhibiting colony formation initiated by single cells.100 cells are plated in 60 mm dishes and incubated drug conjugates undervarious conditions, and allowed the singles cells to form colonies overa period of 7-10 days. Cells are rinsed and stained with methylene blue.Cell colonies (>20 cells) are counted using an AcuCount1000 (Bio Logics)counter.

LDH Release Assay.

This is based on the determination of cell membrane integrity and theleakage of LDH from dead cells. Cells are plated in 96 wells and exposedto drug conjugates for varying times. The conjugates are removed andincubated with a fluorogenic substrate for LDH (“Cyto Tox-One”,Promega). The generated fluorescence is measured (excitation 560 nm andemission 590 nm) as a function of the concentration of LDH in the media.

Apoptosis Sensing Assay.

If needed, in order to differentiate between apoptosis vs. necrosis,apoptosis-sensing assays such as “Annexin V-PS staining” (BD Sciences)and “CaspaTag” Caspase 3 binding (Chemicon) assays are conducted.

Example 24 Demonstrate Release of Morphine/Hydromorphone and NaloxoneCompounds in vivo at Concentrations and Conditions Required for ClinicalEffect

Methods were designed to evaluate the release kinetics and activity ofdrugs, prodrugs, and conjugated drugs with dendrimers. This is done todocument that both the specific activity and dosage of drug achievablewith these systems are adequate for the desired therapeutic and feedbackeffects. These studies are performed with parallel development of boththe Morphine/Hydromorphone and Naloxone arms with combination testing asan ongoing process. It is anticipated that a number of additionalcompounds will be created as well as modifications to promisingcandidates in order to arrive at optimal release kinetics. Ultimately,utilizing a down selection process based on efficacy testing and ADMET,the best compounds will be placed in a preclinical simulation providingevidence for an IND submission to the FDA.

The components of each phase possess unique requirements in animal modeldesign and testing which have been considered to enhance our capabilityfor success.

Selection of Animals for Separately Examining Morphine/Hydromorphone andNaloxone Pharmaco-Kinetics.

(a) Hartley Guinea Pigs (see, e.g., Nambiar, M. P., et al., Toxicologyand Applied Pharmacology, 2007. 219(2-3): p. 142-150; Shih, T. M., T. C.Rowland, and J. H. McDonough, Journal of Pharmacology and ExperimentalTherapeutics, 2007. 320(1): p. 154-161; each herein incorporated byreference in their entireties).

Adult male Hartley guinea pigs weighing 500 grams are an accepted modelutilized by the esterase scientific community. Guinea pigs allow forinitial testing of large numbers of prodrug/conjugates which can bestudied in an economical and effective way. In addition, these animalsare adequate size to be intubated under general anesthetic conditionsfor airway control and hypoxia induction. Invasive hemodynamicmonitoring is required to validate hypoxic release of Naloxone is alsopossible in this animal model. Animal size allows for the collection ofapproximately 5 ml whole blood prior to an end terminal bleed whichlimits the number of time points for collection of an otherwise optimalsmall animal model for study.

(b) Alternative Strategy: Esterase Knockout Mouse.

An esterase knockout mouse, bred specifically to simulate in-vivo humanesterase conditions, is provided. This model provides highlytranslatable data to the human condition. This model serves as a backupto guinea pig for Morphine/Hydromorphone release.

In vivo Morphine/Hydromorphone Pharmacokinetics Studies.

A total of 12 pro-drug/pro-drug compounds are tested at 6 dosage levels,utilizing 2 delivery methods, with 6 evaluation time points per animal.Three animals for each group for statistical validity are used. Bloodsamples are drawn at time 0 prior to drug administration and at serialtime points of 10, 30, 60 min and 6, 12, 24 hours via a placedindwelling catheter. For the first 6 time points 0.5 ml are withdrawn.This is followed by an end terminal bleed for approximately 10 ml finalwhole blood. These samples immediately undergo analysis forMorphine/Hydromorphone levels in serum using an ELISA assay. For thebest 2 drugs only, recovery procedures are carried out with continuedblood sampling at 2 weeks and 1 month followed by sacrifice andhistopathology to validate the lack of long-term toxicity.

For serum Morphine/Hydromorphone level determination, the CalbiotechMorphine Specific Direct ELISA Kit is utilized. In 96 well plates, 20 ulserum is incubated per manufacturer instructions. Both positive andnegative controls are analyzed as provided in the assay. The resultingproducts are read utilizing a 96 well plate reader Bio-Rad 680 XRMicroplate Reader and analyzed using provided statistical software. Theassay's reported sensitivity is to 1 ng/mL, which is more than adequatefor the anticipated in-vivo serum levels.

In vivo Naloxone Pharmacokinetics Studies.

Guinea Pigs are anesthetized and the carotid artery accessed forplacement of an invasive oxygen monitor catheter which provides directmeasurement of blood oxygenation during the study. Blood is drawn as apreoperative standard and then 6 prodrug/conjugates are administeredusing 6 different dosage schedules via 2 delivery methods. Hypoxia isthen induced using a gas mixture to obtain the desired levels of Sa02(10, 20, 40, 70, 100). Blood is collected and analyzed at each of theselevels to confirm appropriate drug release at the desired hypoxiclevels. These samples immediately undergo analysis for Naloxone levelsusing HPLC. For the best 2 drugs only, recovery procedures are carriedout with continued blood sampling at 2 weeks and 1 month followed bysacrifice and histopathology to validate the lack of long-term toxicity.

HPLC (see, e.g., Kuracka, L., et al., Clinical Chemistry, 1996. 42(5):p. 756-760; Orlovic, D., et al., Chromatographia, 2000. 52(11/12): p.732-734; Svensson, J., et al., Journal of Chromatography B: BiomedicalSciences and Applications 1982. 230(2): p. 427-432; Svensson, J.-O.,Journal of Chromatography B: Biomedical Sciences and Applications, 1986.375: p. 174-178; Tebbett, I. R., Chromatographia, 1987. 23(5): p.377-378; each herein incorporated by reference in their entireties) isutilized to quantitatively analyze Naloxone content in the serumsamples. Prior to HPLC analysis of the reaction mixtures, the samplesare pre-treated by protein precipitation by organic solvents. If needed,Solid Phase Extraction method carried out by using C₁₈ Sep-pakcartridges to remove proteins and any other interfering matriximpurities is also performed. After pre-treatment, the samples arereconstituted with the HPLC eluent and injected into a reverse phasecolumn. The complete qualitative and quantitative analysis using HPLC iscarried out on a Waters Delta 600 HPLC system equipped with a Waters2996 photodiode array detector, a Waters 717 Plus auto sampler, WatersFraction collector III and Empower 2 software. An analytical size column(C8 or C18) with a particle size of 5 μm is used. Initially, anisocratic elution using acetonitrile/phosphate buffer, pH 7.0 is used.The conditions for an HPLC experiment, if required, are modified inorder to incorporate various analyses and generate efficient results. Todetermine the percent recovery of a drug sample from the serum, astandard drug sample of a known concentration is spiked into serumand/or cerebrospinal fluid. This spiked sample is then subjected tosample pre-treatment. A HPLC analysis is performed on this pre-treatedspiked sample and a standard drug sample. From the calibration curve andthe regression equation, the percent recovery is computed.

For quantitative analysis, standard drug samples are obtained and stocksolutions for each of these drugs made in an appropriate solvent. Usinga serial dilution method, standards for each drug at variousconcentrations are prepared to generate a calibration curve.Concentration of free drug after its release is calculated using aregression equation.

Example 25 Survey ADMET Characteristics of those Compounds that ShowedDesirable Release Kinetics

ADMET data has already been obtained for Morphine/Hydromorphone,Naloxone and the dendrimers themselves. Therefore, toxicity issues fromthese compounds will not be a limiting factor in selection of the finalcompound. Analysis of the final formulations is conducted to assure thatthey are also not toxic. Therefore, ADMET characteristics of only thosecompounds that show desirable release kinetics will need to be surveyed.Three adult male Hartley pigs (500 grams) per compound are used toensure statistical validity. Six dosage levels with 2 delivery methodsare explored with the goal of achieving dose related-toxicity in thehighest group for definition of the therapeutic threshold.Morphine/Hydromorphone, and 2 Naloxone, ¹³C radio-labeled compounds willundergo ADMET testing.

An indwelling port is placed to assist with scheduled blood draws.Following drug administration, blood samples are drawn at serial timepoints of 1, 4, 7, and 14 days. An end terminal bleed is completed andanimals will be sacrificed with 10 organ harvest for histopathologicalexamination to determine distribution and toxicity effects. Bloodsamples undergo HPLC analysis for evaluation of compound/conjugatelevels.

Example 26 Perform in vivo Studies Simultaneously Examining the Effectof the Morphine/Hydromorphone and Naloxone Compounds

A component of developing a controlled analgesic release system isdetermining the mixture of Morphine/Hydromorphone and Naloxone necessaryobtain the desired clinical effects. Due to the fast drug-releasekinetics, pro-drugs are used for the initial loading bolus to reach thedesired drug-serum concentration. Pro-drug dendrimer complexes andconjugates, both of which have shown slower drug release kinetics, areused for maintenance to replace drug lost during the distribution andelimination phases (FIG. 43). Previously published literature (see,e.g., Schulte, H., A. Sollevi, and M. Segerdahl, Pain, 2005. 116(3): p.366-374; Loetsch, J., et al., Clinical Pharmacology and Therapeutics,1996. 60(3): p. 316-325; Hill, H. F., et al., Pain, 1990. 43(1): p.57-67; Hill, H. F., et al., Pain, 1990. 43(1): p. 69-79 each hereinincorporated by reference in their entireties) and commerciallyavailable software are used to guide decisions in determining theappropriate ratios of pro-drug, pro-drug dendrimer complexes andpro-drug dendrimer-conjugates.

Selection of Animal for Separately Examining Morphine/Hydromorphone andNaloxone Pharmacokinetics.

(a) Gottingen Mini-Pigs (see, eg., Worek, F., et al., Toxicology, 2008.244: p. 35-41; herein incorporated by reference in its entirety)

Gottingen pigs are 20 kg and offer a translatable model to humancondition with the ability for both intubation and invasive hemodynamicmonitoring as well as a large quantity of blood for analysis of drugrelease. Data obtained from pigs is adequate for proof of conceptvalidation. This model allows evaluation of numerous time points bothfor Morphine/Hydromorphone release via esterase and Naloxone release viahypoxia.

In vivo Morphine/Hydromorphone and Naloxone Pharmacokinetics Studies.

In vivo studies simultaneously examining the activityMorphine/Hydromorphone- and Naloxone-based compounds is conducted. Dueto the complex monitoring required for these studies, they need to beperformed in Gottingen pigs. Combinations of bothMorphine/Hydromorphone- and Naloxone-based compounds are administered tothe pigs. Pigs are dosed five times (5) over a period of one hour. Thisis done to simulate a potential overdose scenario and therefore enableevaluation of the feedback mechanism within the controlled analgesicrelease system. The pigs are bled regularly for over the course of 12hours.

Gottingen mini pigs are anesthetized and the carotid artery accessed forplacement of an invasive oxygen monitor catheter which provides directmeasurement of blood oxygenation during the study. Blood is drawn as apreoperative standard. Eight combinations of the best 2 drugs from eachcategory (narcotic and anti-narcotic) are evaluated using a clinicalsimulation model applicable to direct in field application. Pigs receiveserial administration of the combination of drugs at time 0, 15, 30, 45,60 min for a total of 5 doses. This simulates a potential overdosescenario and therefore enables evaluation of the feedback mechanismwithin the controlled analgesic release system. If hypoxia has notoccurred following the final dosage, a gas mixture is administered withreduced oxygen concentrations to simulate hypoxia due to analgesicoverdose. Blood samples are taken at 0, 15, 30, 45, 60, and 120 minutes,as well as at SaO₂ values of 10, 20, 40 70, 100. The serum collected isanalyzed using an ELISA assay (Morphine/Hydromorphone) and HPLC(Naloxone) to confirm obtainment of appropriate drug release levels. Forthe 2 most promising compound combinations, recovery procedures arecarried out with continued blood sampling at 2 weeks and 1 month.Following a terminal bleed, the pigs are sacrificed and undergohistopathological examination to determine long-term distribution andtoxicity effects.

Example 27

This example demonstrates in vitro sustained release of morphine using amorphine pro-drug. Pro-drug A (20 μM)

was incubated with porcine liver esterase (Sigma, 0.01 U) in 20 mMphosphate buffer pH 7.0 for 2 and 16 hours. At the end of the timeperiods the samples were frozen at −20° C. and were thawed prior toloading onto the HPLC column. HPLC analysis was performed within 24hours of the incubation. The analysis was performed on a reverse phasecolumn (250×4.6 mm, C5, 300A) with a flow rate of 1 mL/min using agradient elution beginning at 90/10 Water/Acetonitrile with 0.14% TFAand ending at 10/90 within 30 minutes. Released Morphine was monitoredand detected using a PDA detector at wavelength of 280 nm. As shown inFIG. 44, the morphine prog-drug A without esterase is shown withabsorbance starting at 0.000, the morphine pro-drug A with esterase (2hours) is shown with absorbance starting at 0.010, and the morphinepro-drug A with esterase (16 hours) is shown with absorbance starting at0.020.

Example 28

This example demonstrates in vitro release of free morphine from prodrugin different plasma samples. Pro-drug B (250 μM)

was incubated with 50% fresh frozen plasma collected from the indicatedspecies, or with Human Butyryl Choline Esterase (Human BCE, 0.5 U/ml),or with Human Albumin (Sigma, 25 mg/ml) in 50 mM phosphate buffer pH7.0. The samples were incubated at 37° C. and aliquots were withdrawn atthe indicated time-points and the proteins were precipitated with 2volumes of ice-cold 10% DMSO in acetonitrile. The samples weremicro-centrifuged at high speed for 10 minutes at 4° C. and thesupernatants were frozen at −20° C. The samples were thawed prior toloading onto the UPLC column. UPLC analysis was performed within 24hours of the incubation using an Acquity HSS T3 column (2.1×10 mm) Flowrate was maintained at 0.5 ml/min. Released Morphine was monitored usinga PDA detector at 280 nm. The gradient elution used for this methodbegan at 98/2 Water/Acetonitrile containing 0.14% TFA and ended with2/98 Water/Acetonitrile containing 0.14% TFA in 6.5 minutes. The amountof free morphine released was calculated from a standard curve generatedusing different concentrations of morphine subjected to UPLC underidentical conditions. FIG. 45 shows the release kinetics of freemorphine from the prodrug in the various plasma samples.

Example 29

This example demonstrates that naloxone is released from anindolequinone based naloxone prodrug only under low oxygen conditions.Naoxone pro-drug (125 μM) was incubated with 30% fresh frozen humanplasma in 50 mM phosphate buffer pH 7.0 in the presence of 133 μM eachof NADH and NADPH. The reaction mixture was divided into two portions,and to one portion Argon gas was slowly bubbled using a capillary tubeuntil the oxygen pressure reached 18 mm Hg, monitored using a Blood GasAnalyzer. The tube was tightly sealed and transferred into a hypoxiachamber with a pO₂ that was maintained at 18 mm Hg by continuouslypassing Argon gas. The sample was incubated overnight at roomtemperature with the second portion incubated under normoxia conditionkept outside the chamber. At the end of the time period the samples werefrozen at −20° C. and the samples were thawed prior to loading onto theHPLC column. HPLC analysis was performed within 24 hours of theincubation. The analysis was performed on a reverse phase column(250×4.6 mm, C5, 300A) with a flow rate of 1 mL/min using a gradientelution beginning at 90/10 Water/Acetonitrile with 0.14% TFA and endingat 10/90 within 30 minutes. Released Naloxone was monitored and detectedusing a PDA detector at wavelength of 280 nm. FIG. 46 shows thatnaloxone is released from an indolequinone based naloxone prodrug onlyunder low oxygen conditions.

Example 30

This example demonstrates in vivo sustained release of morphine in aguinea pig model. Male Hartley guinea pigs (375-475 g) from Elm Hilllabs were allowed free access to food and water before being used in thetesting of analgesia effects free morphine, morphine complex, andmorphine prodrug A complex

The Randall & Selitto test apparatus (Harvard Instruments) is used tomeasure analgesia and is based on determination of the animal thresholdresponse to pain induced in the paw by the application of a uniformlyincreasing pressure from a conical tip upon the dorsal surface of therear paw which rests on a platform. The weight in grams is taken uponwithdrawal of the animals paw from the platform with five repeatedmeasurements of pain threshold recorded for each time point (control,60, 150, 240, and 360 min) after drug administration. Values arenormalized to control and the mean ±SEM for each time point arerecorded. To reduce tissue damage to the animal a weight limit is usedfor all animals. FIG. 47 shows sustained release of morphine in theguinea pig model over a six hour period with prodrug A.

After collecting the blood samples from the animals, the blood sampleswere spun down and the supernatant (plasma) was taken for furtheranalysis using UPLC (ultra performance liquid chromatography). Prior tosubjecting the samples to UPLC, the samples were pre-treated to removethe plasma proteins using a solid phase extraction protocol. Using aWaters HLB micro elution plates, the samples were passed through thecartridge following a washing and an equilibration step with Water andmethanol respectively, the samples were then loaded on to the cartridgeand followed by elution with 40/60 Acetonitrile/Isopropanol. The solventwas evaporated over night and the samples were reconstituted in UPLCeluent for analysis. UPLC analysis was performed using an Acquity HSS T3column (2.1×10 mm) Flow rate was maintained at 0.5 ml/min. ReleasedMorphine was monitored using a PDA detector at 280 nm. The gradientelution used for this method began at 98/2 Water/Acetonitrile containing0.14% TFA and ended with 2/98 Water/Acetonitrile containing 0.14% TFA in6.5 minutes. FIG. 48 shows in vivo studies with a guinea pig modeldemonstrating that naloxone is release from naloxone—pro-drug only underlow oxygen conditions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the presentinvention.

1. A composition comprising a dendrimer linked to a moiety comprising atrigger agent, a linkage agent, a targeting agent, and at least onetherapeutic agent, wherein said therapeutic agent is selected from thegroup consisting of a pain relief agent designed to reduce and/oreliminate pain in a subject and a pain relief agent antagonist.
 2. Thecomposition of claim 1, wherein said dendrimer is selected from thegroup consisting of a polyamideamine (PAMAM) dendrimer, apolypropylamine (POPAM) dendrimer, and a PAMAM-POPAM dendrimer.
 3. Thecomposition of claim 1, wherein the linkage agent comprises a spacercomprising between 1 and 8 straight or branched carbon chains, whereinsaid straight or branched chains are selected from the group consistingof i) unsubstituted, and ii) substituted with alkyls.
 4. The compositionof claim 1, wherein said dendrimer is acetylated.
 5. The composition ofclaim 1, wherein said trigger agent is configured for one or more of thefollowing: a) to delay release of said pain relief agent from saidmoiety, wherein said trigger agent is an ester bond; b) toconstitutively release said therapeutic agent from said moiety, whereinsaid trigger agent is selected from the group consisting of an amidebond and an ether bond; c) to release said therapeutic agent from saidmoiety under conditions of acidosis; d) to release said therapeuticagent from said moiety under conditions of hypoxia, wherein said triggeragent is selected from the group consisting of indoquinones,nitroheterocyles, and nitroimidazoles; e) to release said therapeuticagent from said moiety in the presence of a brain enzyme, wherein saidtrigger agent is indolequinone, wherein said brain enzyme is diaphorase;f) to permit said composition to cross the blood brain barrier, whereinsaid targeting agent is transferrin; and g) to permit said compositionto bind with a neuron within the central nervous system, wherein saidtargeting agent is a synthetic tetanus toxin fragment, wherein saidsynthetic tetanus toxin fragment comprises an amino acid peptidefragment, wherein said amino acid peptide fragment is HLNILSTLWKYR (SEQID NO:1).
 6. The composition of claim 1, wherein said moiety furthercomprises a locking agent, wherein activation of said locking agentprevents transfer of said composition across the blood brain barrier,wherein said locking agent is selected from the group consisting of i) apyridinium molecule, wherein said pyridinium molecule is activated byenzymes specific to the central nervous system, and ii) a re-dox system,wherein said re-dox system is the 1,4-dihydrotrigonelline⇄trigonelline(coffearine) re-dox system, wherein conversion of lipophilic 1,4-dihydroform (L) in vivo to the hydrophilic quaternary form (L⁺) by oxidationprevents said composition from diffusing across the blood brain barrier.7. The composition of claim 1, wherein said pain relief agent isselected from the group consisting of: i) an analgesic drug, whereinsaid analgesic drug is selected from the group consisting of: a)non-steroidal anti-inflammatory drugs selected from the group consistingof Acetylsalicylic acid (Aspirin), Amoxiprin, Benorylate/Benorilate,Choline magnesium salicylate, Diflunisal, Ethenzamide, Faislamine,Methyl salicylate, Magnesium salicylate, Salicyl salicylate,Salicylamide, arylalkanoic acids, Diclofenac, Aceclofenac, Acemethacin,Alclofenac, Bromfenac, Etodolac, Indometacin, Nabumetone, Oxametacin,Proglumetacin, Sulindac, Tolmetin, 2-arylpropionic acids, Ibuprofen,Alminoprofen, Benoxaprofen, Carprofen, Dexibuprofen, Dexketoprofen,Fenbufen, Fenoprofen, Flunoxaprofen, Flurbiprofen, Ibuproxam,Indoprofen, Ketoprofen, Ketorolac, Loxoprofen, Naproxen, Oxaprozin,Pirprofen, Suprofen, Tiaprofenic acid), N-arylanthranilic acids,Mefenamic acid, Flufenamic acid, Meclofenamic acid, Tolfenamic acid,pyrazolidine derivatives, Phenylbutazone, Ampyrone, Azapropazone,Clofezone, Kebuzone, Metamizole, Mofebutazone, Oxyphenbutazone,Phenazone, Sulfinpyrazone, oxicams, Piroxicam, Droxicam, Lornoxicam,Meloxicam, Tenoxicam, sulphonanilides, nimesulide, licofelone, andomega-3 fatty acids; b) COX-2 inhibitors selected from the groupconsisting of Celecoxib, Etoricoxib, Lumiracoxib, Parecoxib, Rofecoxib,and Valdecoxib; and c) opiate drugs selected from the group consistingof natural opiates, alkaloids, morphine, codeine, thebaine,semi-synthetic opiates, hydromorphone, hydrocodone, oxycodone,oxymorphone, desomorphine, diacetylmorphine (Heroin), nicomorphine,dipropanoylmorphine, diamorphine, benzylmorphine, Buprenorphine,Nalbuphine, Pentazocine, meperidine, diamorphine, ethylmorphine, fullysynthetic opioids, fentanyl, pethidine, Oxycodone, Oxymorphone,methadone, tramadol, Butorphanol, Levorphanol, propoxyphene, endogenousopioid peptides, endorphins, enkephalins, dynorphins, and endomorphinsii) an anxiolytic drug selected from the group consisting ofbenzodiazepines, alprazolam, bromazepam (Lexotan), chlordiazepoxide(Librium), Clobazam, Clonazepam, Clorazepate, Diazepam, Midazolam,Lorazepam, Nitrazepam, temazepam, nimetazepam, Estazolam, Flunitrazepam,oxazepam (Serax), temazepam (Restoril, Normison, Planum, Tenox, andTemaze, Triazolam, serotonin lA agonists, Buspirone (BuSpar),barbituates , amobarbital (Amytal), pentobarbital (Nembutal),secobarbital (Seconal), Phenobarbital, Methohexital, Thiopental,Methylphenobarbital, Metharbital, Barbexaclone), hydroxyzine,cannabidiol, valerian, kava (Kava Kava), chamomile, Kratom, Blue Lotusextracts, Sceletium tortuosum (kanna) and bacopa monniera; iii) ananesthetic drug selected from the group consisting of local anesthetics,procaine, amethocaine, cocaine, lidocaine, prilocaine, bupivacaine,levobupivacaine, ropivacaine, dibucaine, inhaled anesthetics,Desflurane, Enflurane, Halothane, Isoflurane, Nitrous oxide,Sevoflurane, Xenon, intravenous anesthetics, Barbiturates, amobarbital(Amytal), pentobarbital (Nembutal), secobarbital (Seconal),Phenobarbital, Methohexital, Thiopental, Methylphenobarbital,Metharbital, Barbexaclone)), Benzodiazepines, alprazolam, bromazepam(Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam,Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam,nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam, Etomidate,Ketamine, and Propofol; iv) an antipsychotic drug selected from thegroup consisting of butyrophenones, haloperidol, phenothiazines,Chlorpromazine (Thorazine), Fluphenazine (Prolixin), Perphenazine(Trilafon), Prochlorperazine (Compazine), Thioridazine (Mellaril),Trifluoperazine (Stelazine), Mesoridazine, Promazine, Triflupromazine(Vesprin), Levomepromazine (Nozinan), Promethazine (Phenergan)),thioxanthenes, Chlorprothixene, Flupenthixol (Depixol and Fluanxol),Thiothixene (Navane), Zuclopenthixol (Clopixol & Acuphase)), clozapine,olanzapine, Risperidone (Risperdal), Quetiapine (Seroquel), Ziprasidone(Geodon), Amisulpride (Solian), Paliperidone (Invega), dopamine,bifeprunox, norclozapine (ACP-104), Aripiprazole (Abilify),Tetrabenazine, and Cannabidiol; v) a hypnotic drug selected from thegroup consisting of Barbiturates, Opioids, benzodiazepines, alprazolam,bromazepam (Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam,Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam,nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam,nonbenzodiazepines, Zolpidem, Zaleplon, Zopiclone, Eszopiclone,antihistamines, Diphenhydramine, Doxylamine, Hydroxyzine, Promethazine,gamma-hydroxybutyric acid (Xyrem), Glutethimide, Chloral hydrate,Ethchlorvynol, Levomepromazine, Chlormethiazole, Melatonin, and Alcohol;vi) a sedative drug selected from the group consisting of barbituates,amobarbital (Amytal), pentobarbital (Nembutal), secobarbital (Seconal),Phenobarbital, Methohexital, Thiopental, Methylphenobarbital,Metharbital, Barbexaclone), benzodiazepines, alprazolam, bromazepam(Lexotan), chlordiazepoxide (Librium), Clobazam, Clonazepam,Clorazepate, Diazepam, Midazolam, Lorazepam, Nitrazepam, temazepam,nimetazepam, Estazolam, Flunitrazepam, oxazepam (Serax), temazepam(Restoril, Normison, Planum, Tenox, and Temaze), Triazolam, herbalsedatives, ashwagandha, catnip, kava (Piper methysticum), mandrake,marijuana, valerian, solvent sedatives, chloral hydrate (Noctec),diethyl ether (Ether), ethyl alcohol (alcoholic beverage), methyltrichloride (Chloroform), nonbenzodiazepine sedatives, eszopiclone(Lunesta), zaleplon (Sonata), zolpidem (Ambien), zopiclone (Imovane,Zimovane)), clomethiazole (clomethiazole), gamma-hydroxybutyrate (GHB),Thalidomide, ethchlorvynol (Placidyl), glutethimide (Doriden), ketamine(Ketalar, Ketaset), methaqualone (Sopor, Quaalude), methyprylon(Noludar), and ramelteon (Rozerem); and vii) a muscle relaxant drugselected from the group consisting of depolarizing muscle relaxants,Succinylcholine, short acting non-depolarizing muscle relaxants,Mivacurium, Rapacuronium, intermediate acting non-depolarizing musclerelaxants, Atracurium, Cisatracurium, Rocuronium, Vecuronium, longacting non-depolarizing muscle relaxants, Alcuronium, Doxacurium,Gallamine, Metocurine, Pancuronium, Pipecuronium, and d-Tubocurarine. 8.The composition of claim 1, wherein said pain relief agent antagonist isa drug that counters the effect of a pain relief agent, wherein saidpain relief agent antagonist is selected from the group consisting of ananesthetic antagonist, an analgesic antagonist, a mood stabilizerantagonist, a psycholeptic drug antagonist, a psychoanaleptic drugantagonist, a sedative drug antagonist, a muscle relaxant drugantagonist, and a hypnotic drug antagonist.
 9. The composition of claim1, wherein said pain relief agent antagonist is selected from the groupconsisting of a respiratory stimulant, Doxapram, BIMU-8, CX-546, anopiod receptor antagonist, Naloxone, naltrexone, nalorphine,levallorphan, cyprodime, naltrindole, norbinaltorphimine,buprenorphine), a benzodiazepine antagonist, flumazenil, anon-depolarizing muscle relaxant antagonist, and neostigmine.
 10. Thecomposition of claim 1, wherein said moiety comprises two therapeuticagents and two pain relief agents.
 11. The composition of claim 10,wherein said two relief agents are ketamine and lorazepam.
 12. Thecomposition of claim 1, wherein said therapeutic agent is a pain reliefagent, wherein said pain relief agent is morphine.
 13. The compositionof claim 1, wherein said therapeutic agent is a pain relief agentantagonist, wherein said pain relief agent antagonist is Doxapram. 14.The composition of claim 1, wherein said therapeutic agent is a painrelief agent antagonist, wherein said pain relief agent antagonist isNaloxone.
 15. A method of reducing pain in a subject comprisingadministering to a subject at least one composition of claim
 1. 16. Themethod of claim 15, wherein said subject is a human.
 17. The method ofclaim 15, wherein two compositions are administered to said subject suchthat one of said compositions comprises a pain relief agent and one ofsaid compositions comprises a pain relief agent antagonist.
 18. Themethod of claim 17, wherein said pain relief agent is morphine, whereinsaid pain relief agent antagonist is Naloxone.
 19. The method of claim17, wherein said pain relief agent is ketamine, wherein said pain reliefagent antagonist is Doxapram.
 20. The method of claim 17, wherein saidpain relief agent is lorazepam, wherein said pain relief agentantagonist is Doxapram.